Characterization and detection of sequences associated with autoimmune diseases

ABSTRACT

DNA sequences and corresponding amino acid sequences from the HLA class II beta region of the human genome that are associated with insulin-dependent diabetes mellitus (IDDM) and Pemphigus vulgaris (PV) have been identified. Specifically, marker DNA sequences which detect either directly or indirectly the identity of the codon encoding for the amino acid at position 57 of the DQβ protein sequence are disclosed as well as sequences from the DRβ region. These sequences may be used to generate DNA hybridization probes and antibodies for assays to detect a person&#39;s susceptibility to autoimmune diseases, such as IDDM and PV. Such antibodies and peptides encoded by said DNA sequences can be used therapeutically or prophylactically.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 07/121,519,filed Nov. 17, 1987, which is a continuation-in-part application of nowabandoned Ser. No. 06/899,512 filed Aug. 22, 1986 and of now abandonedSer. No. 06/899,344 filed Aug. 22, 1986, which is a continuation-in-partapplication of now abandoned Ser. No. 839,331 filed Mar. 13, 1986.

BACKGROUND OF THE INVENTION

A variety of autoimmune diseases have been associated with serologicallydefined variants of the human leukocyte antigen (HLA) class IIIantigens. The HLA region, located on the short arm of chromosome 6,encodes many different glycoproteins that have been classified into twocategories. The first category, class I products, encoded by the HLA-A,-B, and -C loci, are on the surface of all nucleated cells and functionas targets in cytolytic T-cell recognition. The second category, classII products, encoded by the HLA-D region, are involved in cooperationand interaction between cells of the immune system. These class IIproducts appear to be encoded by at least three distinct loci, DR, DQand DP, each with its distinct alpha and beta chains. The class II lociof the human major histocompatibility complex (MHC) encode highlypolymorphic cell-surface glycoproteins (macrophage and β-celltransmembrane glycoproteins). [For review article, see Giles et al. Adv.in Immunol. 37:1-71 (1985).] The polymorphism in class II antigens islocalized to the NH₂ -terminal outer domain and is encoded by the secondexon. The class II polymorphic residues have been postulated to interactwith the T-cell receptor or with foreign antigen or both [Sette et al.,Nature, 328:395-399 (1987)] with recognition of the antigen peptidefragments in association with a specific class II product leading to Tcell activation and consequent stimulation of antibody production by βlymphocytes [Marx et al., Science, 238:613-614 (1987).]

This invention relates to HLA class II beta genes and proteinsassociated with autoimmune diseases and methods for their diagnosticdetection. Specifically, the autoimmune diseases on which this inventionfocuses are insulin-dependent diabetes mellitus (IDDM) and Pemphigusvulgaris (PV).

Insulin-dependent diabetes mellitus (IDDM), a chronic autoimmune diseasealso known as Type I diabetes, is a familial disorder of glucosemetabolism susceptibility associated with specific allelic variants ofthe human leukocyte antigens (HLA). The dysfunctional regulation ofglucose metabolism occurring in IDDM patients results from theimmunologically mediated destruction of the insulin-producing isletcells of the pancreas, the beta cells. The development of IDDM can bedivided into six stages, beginning with genetic susceptibility andending with complete destruction of beta-cells. G. Eisenbarth, N. Eng.J. Med., 314:1360-1368 (1986). [Donaich et al., Annu. Rev. Med.,34:13-20 (1983).]. More than 90% of all IDDM patients carry the DR3and/or DR4 antigen, and individuals with both DR3 and DR4 are at greaterrisk than individuals who have homozygous DR3/3 or DR4/4 genotypes. L.Raffel and J. Rotter, Clinical Diabetes., 3:50-54 (1985); Svejgaard etal., Immunol. Rev., 70:193-218 (1983); L. Ryder et al., Ann. Rev.Genet., 15:169-187 (1981).

Pemphigus derived from the Greek pemphix meaning blister or pustule isthe name applied to a distinctive group of chronic or acute skindiseases characterized by successive crops of itching bullae. Pemphigusvulgaris (PV) is a rare relapsing disease manifested by suprabasal,intraepidermal bullae of the skin and mucus membranes, which isinvariably fatal if untreated; however, remission has been obtained bythe use of corticosteroid hormones and immunosuppressive drugs. PV, anautoimmune disease, has been strongly associated with the HLA serotypesDR4 and DRw6 [Brautbar et al., Tissue Antigens, 16:238-241 (1986)] withless than 5% of PV patients possessing neither marker. Diseaseassociations with two different haplotypes can be interpreted to mean(1) the two haplotypes share a common allele or epitope, or,alternatively, that (2) different alleles on the two haplotypes arecapable of conferring disease susceptibility.

Molecular analysis of the HLA class II genes has revealed that the HLAserotypes are genetically heterogeneous, and that, in particular, theDR4 haplotype consists of five different DRβI allelic sequencescorresponding to the five mixed lymphocyte culture (MLC) defined typesDw4, Dw10, Dw13, Dw14 and Dw15 [Gregersen et al., PNAS (U.S.A.)83:2642-2646 (1986)] and three different DQβ allelic sequencescorresponding to the DQβ3.1, DQβ3.2 and DQ-blank types [Erlich et al.,in Schacter et al. (eds.): The Molecular Analysis of HistocompatibilityAntigens, pp. 93-109 (1987)]. Virtually all of the extensivepolymorphism characteristic of the class II loci has been localized tothe second exon.

Sequence analysis of coding sequence polymorphisms in the DRβ locirevealed that the sequence or epitope in the DR4 DRβI chain thatdistinguishes Dw10 from the other DR4 subtypes is shared by the DRβIchain of the DRw6 haplotype. [Gorski et al., Nature, 322:67-70 (1986)].Recently, restriction fragment length polymorphisms (RFLPs) whichsubdivide the DR4 and DRw6 haplotypes were obtained by using a HLA-DQβcDNA probe; such RFLPs have been reported to be even more highlyassociated with PV than are the serologic markers [Szafer et al., 1987,Proc. Natl. Acad. Sci. USA 84: 6542-6545].

Of all the immunologically defined polyomorphisms, the HLA-DR betaregion has been found to be most strongly associated with IDDM.Therefore, restriction fragments of the HLA class II-β DNA have beenanalyzed for use as genetic markers of insulin-dependent diabetesmellitus. D. Owerbach et al. Diabetes, 33:985-964 (1984); O.Cohen-Haguenauer et al., PNAS (U.S.A.), 82:3335-3339 (1985); D. Stetleret al., PNAS (U.S.A.), 82:8100-8104 (1985).

Arnheim et al., PNAS (U.S.A.), 82: 6970-6974 (October 1985), examinedDNA polymorphisms within the HLA class II loci associated withsusceptibility to IDDM by using genomic blot-hybridization analysis withDQβ and DRβ cDNA probes. Described therein is a DQβ subdivision of theDR4 haplotype wherein one DR4 variant has a RsaI restriction fragmentlength polymorphism (RFLP) of 1.8 kb and another had a RsaI RFLP of 1.5kb. The DQβ-related 1.5 kb RsaI fragment was reported to identify anumber of non-DR4 IDDM individuals as well as 90% of all IDDM DR4individuals.

Other investigators using other restriction enzymes (e.g., BamHI,HindIII) have reported RFLP subdivisions of the DR4 haplotype using DQβprobes. [Holbeck et al., Immunogenetics (1986) 24:251-258, Henson etal., Immunogenetics, (1987) 25:152-160).] Holbeck et al., id., foundthat the RFLP subsets of DR4, designated DQw3.1 and DQw3.2 aredistinguishable by the reactivity of their expressed products with aspecific monoclonal antibody TA10. [Kim et al., PNAS (U.S.A.),82:8139-8142 (1985); Tait et al., Tissue Antigens, (1986) 28:65-71.] TheDQw3.1 subtype correlates with the serologic specificity TA10⁺, whereasDQw3.2 correlates with TA10⁻.

U.S. Ser. No. 899,344 (filed Aug. 22, 1986) entitled "Process forDetecting Specific Nucleotide Variations and Genetic PolymorphismsPresent in Nucleic Acids" (incoporated herein by reference) disclosesthe cloning and sequencing of the RsaI 1.5 kb (DQw3.2) and the RsaI 1.8kb (DQw3.1) variants of DR4 haplotypes and illustrates the differencesin the sequences thereof. (Such differences are shown herein in TablesIII and IV.)

WO 86/07464 discloses a specific DQβ₂ allelic variant, DQw3.2 as aspecific genomic marker associated with IDDM, and provides two methodsof identifying individuals at increased risk of diabetes. The firstmethod involves the use of a labeled probe to detect the DQw3.2 allele,whereas the second method involves the serologic detection of the DQw3.2allele.

Erlich et al., in Schacter et al., (eds.), Perspectives inImmunogenetics and Histocompatibility, Vol. 7:93-106 (1987), reportedthe protein translation sequences for the DQw3.1 and 3.2 variants.

Michelson et al., J. Clin. Invest., 79:1144-1152 (April 1987), reportedthe nucleotide sequence for the DQw3.1 variant.

Acha-Orbea et al., PNAS (U.S.A.), 84(8): 2435-2435 [1987) reported ondifferences in the H-2 I-A region of control mice anddiabetes-susceptible NOD (non-obese diabetic) mice. Normal mice have anaspartate residue at position 57 of said region whereas NOD mice have aneutral serine residue at that position. The human HLA-DQβ region isanalogous to the H-2 I-A region of the mouse.

Yoon et al., Diabetes Care, 8 (suppl. 1):39-44 (Sept.-Oct. 1985),presents a review of the evidence for viruses as a trigger for IDDM inanimals and humans. See also, Bodansky et al., Lancet, (1986),ii:1351-1353; Kagnoff et al., J. Exp. Med., (1984), 160:1544-1557;McChesney et al., Ann. Rev. Immunol. (1987), 5:279-304; Oldstone et al.,in Notkins et al. (eds.), Concepts in Viral Pathogenesis (1986);Schwimmback et al., J. Exp. Med. (1987), 166:173-181; Silver et al.,Disease Markers (1985), 3:155-168; and Srinivasappa et al., J. Virol.,57:397-401.

Roudier et al., Abstract from the American Rheumatism Association(Western Region) meeting in San Diego, Calif., Nov. 5-7, 1987, reportedthat the HLA Dw4 DRβ1 chain and an Epstein-Barr virus (EBV) glycoproteinshare a hexapeptide.

Todd et al., Nature, 329:599-604 (Oct. 15, 1987) discusses thecontribution of the HLA DQβ gene to susceptibility and resistance toIDDM. The authors conclude that "the structure of the DQ molecle, inparticular residue 57 of the β-chain, specifies the autoimmune responseagainst the insulin-producing islet cells."

Many HLA DRβ sequences have been published previously. The sequenceAspIleLeuGluAspGluArg was reported by Gregersen et al., PNAS (U.S.A.),83:2642-2646 (1986) as part of a study of the diversity of DRβ genesfrom HLA DR4 haplotypes. No mention was made of an association thereofwith diabetes. In addition, J. Gorski and B. Mach, Nature, 322:67-70(1986) reported on HLA-DR polymorphism within a group including thehaplotypes DR3, DR5 and DRw6. The nucleotide sequences found in thepolymorphic regions at the βI locus were not discussed regardingassociation with diabetes. The first publication on HLA sequences fromdiabetics is that by D. Owerbach et al., Immunogenetics, 24:41-46(1986). This paper is based on the study on a HLA-DRβ gene library fromone IDDM patient. The analysis of class II polymorphism and diseasesusceptibility requires the comparison of many sequences derived frompatients and HLA-matched controls.

Allelic variation in the class II antigens is restricted to the outerdomain encoded by the second exon of the protein. Serologic methods fordetecting HLA class II gene polymorphism are not capable of detectingmuch of the variation detectable by DNA methods.

Allelic variations may be detected independently of restriction sitepolymorphism by using sequence-specific synthetic oligonucleotideprobes. Conner et al., PNAS (U.S.A.), 80:278 (1983). This technique hasbeen applied to study the polymorphism of HLA DR-β using Southernblotting. Angelini et al., PNAS (U.S.A.), 83:4489-4493 (1986).

A further refinement of the technique using sequence-specificoligonucleotide probes involves amplifying the nucleic acid sample beinganalyzed using selected primers, four nucleotide triphosphates, and anappropriate enzyme such as DNA polymerase, followed by detecting thenucleotide variation in sequence using the probes in a dot blot format,as described in now abandoned U.S. Ser. No. 899,344, supra, and in nowabandoned Ser. No. 839,331 filed Mar. 13, 1986. A temperature cyclingprocess wherein a thermostable enzyme is added only once in theamplification process is described in now abandoned U.S. Ser. Nos.899,513 and copending 063,647 filed respectively Aug. 22, 1986 and Jun.17, 1987, both entitled "Process for Amplifying, Detecting, and/orCloning Nucleic Acid Sequences Using a Thermostable Enzyme." Nowabandoned U.S. Ser. No. 899,241 and U.S. Pat. No. 4,889,818 respectivelyAug. 22, 1986 and Jun. 17, 1987, both entitled "Purified ThermostableEnzyme" disclose and claim thermostable enzymes, purified orrecombinant, which can be used in said amplification process.

There is a need in the art for subdivision of the serologic markers HLADR3, DR4 and DRw6 to obtain more information and more precisely definedmarkers for susceptibility to the autoimmune diseases IDDM and PV.Further, there is a need in the art to identify susceptibilityconferring haplotypes which are neither DR3, DR4 nor DRw6.

Previously, the distinction between the IDDM associated DQβ variants,DQw3.1 and DQw3.2, of the DR4 haplotype has been made by RFLP or by theuse of antibodies. This invention in one aspect relates to methods toidentify such DQβ variants.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a marker DR beta-I DNAsequence from the HLA class II beta genes associated withinsulin-dependent diabetes mellitus (IDDM) and with IDDM and withDR4-associated susceptibility to Pemphigus vulgaris (PV).

Specifically, in one aspect, the present invention provides a marker DRbeta-I DNA sequence associated with IDDM and with DR4-associatedsusceptibility to Pemphigus vulgaris which is GACATCCTGGAAGACGAGCGG, orthe DNA strand complementary thereto. Further, the invention providesfor the amino acid sequence Asp Ile Leu Glu Asp Glu Arg which is encodedby said DNA sequence.

The present invention also provides for marker DR beta-I DNA sequencesassociated with DR4, Dw4-associated susceptibility to IDDM wherein onesuch sequence is GGAGCAGAAGCGGGCCGCG, or the DNA strand complementarythereto. Further, the invention concerns amino acid sequences encoded bysaid marker DR beta-I DNA sequences associated with DR4, Dw4-associatedsusceptibility to IDDM and antibodies to said amino acid sequences.

The invention further provides for marker DQβ DNA sequences from the HLAclass II beta genes associated with DRw6-associated susceptibility toPemphigus vulgaris. Specifically, such DQβ DNA sequences comprise one ormore nucleotide sequences from the second exon of the unique DQB1.3allele from about codon 20 to about codon 80. Further, the inventionconcerns amino acid sequences encoded by such DQB1.3 DNA sequences andantibodies to said amino acid sequences.

In another aspect, the invention provides for a marker DR3 beta-III DNAsequence from the HLA class II beta genes associated withinsulin-dependent diabetes mellitus selected from the group consistingof:

(a) GAGCTGCGTAAGTCTGAG,

(b) GAGGAGTTCCTGCGCTTC, and

(c) CCTGTCGCCGAGTCCTGG,

or the DNA strands which are complementary thereto.

In still another aspect, the invention provides an amino acid sequencefrom HLA class II beta region of the human genome associated withinsulin-dependent diabetes mellitus which forms a peptide selected fromthe group consisting of:

(a) Glu Leu Arg Lys Set Glu,

(b) Glu Glu Phe Leu Arg Phe, and

(c) Pro Val Ala Glu Ser Trp.

In another aspect, the invention provides a marker DNA sequence from theHLA DQ-beta allele associated with susceptibility to insulin-dependentdiabetes mellitus (IDDM), wherein said sequence can be used to detecteither directly or indirectly the identity of the codon at position 57of the DQ-beta protein sequence and marker DNA sequences wherein saidcodon at position 57 is selected from the group consisting of codons foralanine, valine and aspartate. Said marker DNA sequence is preferablyselected from the group consisting of:

(a) GGGCCGCCTGCCGCC,

(b) GGGCTGCCTGCCGCC,

(c) GGGCGGCCTGTTGCC,

(d) GGGCCGCCTGACGCC, and

(e) GGGCGGCCTGATGCC,

or the DNA strands which are complementary thereto.

This invention further relates to oligonucleotide probes specific forthe 1.5 kb (DQβ3.2) variant and for the 1.8 kb (DQβ3.1) variant DQβsubdivisions of the DR4 haplotype. Specifically, such an oligonucleotideprobe specific for the 1.5 kb (Dqβ3.2) varient is designated GH74, is a19-mer and has the sequence:

CCGCTGGGGCCGCCTGCCG.

An oligonucleotide probe, designated GH92 specific for the 1.8 (DQβ3.1)RFLP is also a 19-mer and has the following sequence:CGTGGAGGTGTACCGGGCG. Under appropriate hybridization and washingconditions, these oligonucleotides, labeled, for example, with ³² P bykinasing, or with non-radioisotopic molecule reporters, such as biotinor an enzyme as, for example, horseradish peroxidase, specificallyidentify the DQβ3.2 and DQβ3.1 variants. This invention further relatesto the diagnostic use of such probes.

Further, the invention provides for marker DNA sequences wherein saidDNA sequences are used to detect indirectly a second DNA sequencecomprising the codon at position 57 wherein said codon₅₇ is selectedfrom the group consisting of codons for alanine, valine and aspartate.Said marker DNA sequences used to detect indirectly said second DNAseqence, are preferably selected from the group consisting of:

(a) GTGGGGGTGTATCGGGCG,

(b) GTGGGGGAGTTCCGGGCG,

(c) GTGGAGGTGTACCGGGCG, and

(d) GTGGGGGTGTACCGGGCA,

or the DNA strands which are complementary thereto.

Further, the invention provides for allele-specific oligonucleotide(ASO) probes that can be used to detect indirectly the identity of codon57 in the DQβ locus wherein such probes are preferably selected from thegroup consisting of:

(1) a 19-mer designated GH61 from the DQ-beta-B region of the DR3haplotype having the nucleotide sequence: CGGCAGGCAGCCCCAGCAG;

(2) a 19-mer designated GH66 from the DQα region of the DR3 haplotypehaving the nucleotide sequence: TGTTTGCCTGTTCTCAGAC; and

(3) a 21-mer designated GH70 from the DQ-beta-A region of the DR3haplotype having the nucleotide sequence: GATGCTTCTGCTCACAAGACG.

The invention also relates to a process for detecting the presence orabsence of sequences associated with susceptibility to insulin-dependentdiabetes mellitus and/or Pemphigus vulgaris in a DNA sample comprising:

(a) treating the sample to expose the DNA therein to hybridization;

(b) affixing the treated sample to a membrane;

(c) treating the membrane under hybridization conditions with a labeledsequence-specific oligonucleotide probe capable of hybridizing with oneor more of the DNA sequences selected from the group consisting of:

(1) GAGCTGCGTAAGTCTGAG,

(2) GAGGAGTTCCTGCGCTTC,

(3) CCTGTCGCCGAGTCCTGG,

(4) GACATCCTGGAAGACGAGCGG,

(5) GGGCCGCCTGCCGCC,

(6) GGGCTGCCTGCCGCC, and

(7) GGGCGGCCTGTTGCC,

or with the DNA strands complementary thereto; and

(d) detecting whether the probe has hybridized to any DNA in the sample.

Still further, the invention concerns an antibody that binds to one ormore of the amino acid sequences selected from the group consisting of:

(a) Glu Leu Arg Lys Ser Glu,

(b) Glu Glu Phe Leu Arg Phe,

(c) Pro Val Ala Glu Ser Trp, and

(d) Asp Ile Leu Glu Asp Glu Arg.

The invention also concerns an antibody that binds to a peptide segmentcontaining an epitope comprising an amino acid residue corresponding toposition 57 of a DQ-beta protein, wherein said antibody may havecross-reactivity with a homologous peptide sequence encoded by a humanpersistent viral pathogen, and wherein said amino acid residue isselected from the group consisting of alanine and valine. Said antibodypreferably binds to a peptide selected from the group consisting of:

(a) Gly Pro Pro Ala Ala,

(b) Gly Leu Pro Ala Ala, and

(c) Gly Arg Pro Val Ala.

Further, said viral pathogen is preferably selected from the group ofviruses consisting of Epstein-Barr virus, rubella virus, Coxsackievirus, cytomegalovirus, and reovirus.

Still further, the invention relates to a process for detecting thepresence or absence of sequences associated with susceptibility toinsulin-dependent diabetes mellitus in a protein sample comprising:

(a) incubating the sample in the presence of one or more of theantibodies that bind to a peptide selected from the group consisting of:

Gly Pro Pro Ala Ala,

Gly Leu Pro Ala Ala, or

Gly Arg Pro Val Ala;

wherein said antibodies are labeled with a detectable moiety; and

(b) detecting the moiety. The antibodies can be polyclonal ormonoclonal. Said process for detecting IDDM associated sequencesincludes those processes wherein before, during, or after incubatingwith the labeled antibody, the sample is incubated in the presence of amonoclonal antibody that is immobilized to a solid support and binds toone or more of the amino acid sequences selected from the groupconsisting of:

(a) Gly Pro Pro Ala Ala,

(b) Gly Leu Pro Ala Ala, and

(c) Gly Arg Pro Val Ala.

The invention further concerns a process for identifying haplotypesassociated with susceptibility to insulin-dependent diabetes mellitus ina serum sample comprising:

(a) incubating the sample in the presence of one or more of the peptidesselected from the group consisting of Gly Pro Pro Ala Ala, Gly Leu ProAla Ala, and Gly Arg Pro Val Ala;

(b) detecting the presence of immune complexes formed between saidpeptide and an antibody present in said serum sample; and

(c) determining from the results of step (b) whether a susceptiblehaplotype is present. The peptides used in said process can be labeledwith a detectable moiety, and the detection can be by enzyme reaction,fluorescence or luminescence emission.

The invention further concerns the prophylactic and therapeutic use ofthe above-referenced antibodies and peptides.

In another aspect, the invention provides a kit for detecting thepresence or absence of sequences associated with susceptibility toinsulin-dependent diabetes mellitus or Pemphigus vulgaris in a DNAsample, which kit comprises, in packaged form, a multicontainer unithaving one container for each labeled sequence-specific DNA probecapable of hybridizing with one or more of the DNA sequences identifiedabove or with the DNA strands complementary thereto.

In a final aspect, the invention provides a kit for detecting thepresence or absence of amino acid sequences associated withsusceptibility to insulin-dependent diabetes mellitus or to Pemphigusvulgaris in a protein sample, which kit comprises, in packaged form, amulticontainer unit having a container for each antibody labeled with adetectable moiety that binds to one or more of the amino acid sequencesidentified above.

As mentioned above, genetic susceptibility to IDDM has been correlatedin both family and population studies with the presence of the serologicmarkers HLA DR3 and DR4. The highest risk for IDDM is associated withHLA DR3,4 heterozygotes, suggesting that the susceptible allelesassociated with these two DR types may be different and that two dosesmay be required for high risk to IDDM. Previous restriction fragmentlength polymorphism analysis has subdivided DR3 and DR4 into two subsetseach.

Similarly, as mentioned above, genetic susceptibility to PV has beencorrelated with the presence of the serologic markers HLA, DR4 and DRw6.Previous restriction fragment length polymorphism has subdivided the DR4and DRw6 haplotypes.

Molecular analyses of the HLA genes herein has resulted in furthersubdivision of the HLA DR3, DR4 and DRw6 serological types and in thegeneration of novel, more informative, and more precisely definedgenetic markers for susceptibility to IDDM and PV. The moleculartechniques herein reveal not only that the number of class II loci isunexpectedly large, but also that the allelic variation at these loci isgreater than the polymorphic series defined by serological typing andcan be more precisely localized.

DESCRIPTION OF FIGURES

FIG. 1 illustrates the sequence of the DRβ fragment PCR (polymerasechain reaction) amplified according to Example III. The sequences of thePCR primers are shown by long arrows, which also indicate the directionof extension by the polymerase. The broken lines show the BamHI and PstIrecognition sequences used to generate restriction sites for cloning.The start of the second exon sequence is shown by the short arrow andthe region of the fragment corresponding to the Dw10 sequence specificoligonucleotide GH78 is shown by the bracketed segment. Digestion withBamHI and PstI produces a 248 bp fragment for cloning. The sequenceshown here represents a prototype DRB1 allele from the DR4 Dw10haplotype.

FIG. 2 illustrates the amino acid sequences for the HLA DRβ second exonfrom three Pemphigus vulgaris (PV) (see Example III) and from DRβprototypes [Gregerson et al., PNAS (U.S.A.), 83:2642-2646 (1986)] usingthe standard one-letter amino acid code (see Table VIII, infra). Theentire amino acid sequence of the DRβ1 allele from a DR4 Dw4 haplotypeis shown whereas the other sequences are aligned with it using a dash toindicate homology and letters to indicate polymorphic amino acids. TheDR type (and the Dw type where appropriate) and the DRB locus assignmentare shown at the right end of each sequence. "I" refers to the DRβ1locus, "III" for the DRβIII locus which encodes the DRw52 specificity,and "IV" for the DRβIV locus, which encodes the DRw53 specificity. Thefragments obtained by PCR cloning are smaller than the prototypesequences, which are derived from cDNA clones.

FIG. 3 illustrates the amino acid sequences from the HLA DRβ second exonof three PV patients according to the conventions of FIG. 2 wherein thesequences are aligned with DR4 DQβ 3.1 and DQβ 3.2 prototype sequencesfrom cDNA clones [Michelsen et al., J. Clin. Invest., 79:1144-1152(1987); Gregersen et al., supra]. At the right of each sequence is theDR type and DQβ type. The DQβ sequences obtained by PCR cloning areshorter than the prototype sequences.

FIGS. 4a and 4b illustrate the alignment of HLA-DQβ protein sequence.DNA sequences (4b is a continuation of 4a) of DQβ alleles weretranslated to the standard one-letter amino acid code (Table VIII,infra) and aligned to show patterns of homology. A dash indicateshomology with the equivalent amino acid in the prototypic DQβ (DR4)allele. A blank indicates that the sequence was not determined. Locationof the PCR amplification primers are shown on the bottom. Note that thePCR amplification procedure only determines the sequence between theoligonucleotide primers [Scharf et al., Science, 233:1076-1078 (1986)].The source of each sequence is designated on the left of each line, andits DR serologic type is shown on the right. An asterisk after the DRtype indicates that the allele was determined from a patient with IDDM.The Hu129 sequence was determined from a PV patient. On the far right isthe designation of the allele, corresponding when possible with the DQwtyping of the haplotype. The DCB4 sequence was from Larhammar et al.,PNAS (U.S.A.), 80:7313-7317 (1983); CMCC and MMCC are from Horn et al.,1988, Pro. Natl. Acad. Sci. USA 85: 60126-6016; DQB37 from Michelsen etal., J. Clin. Invest., 79:1144-1152 (1987); KT3 from Gregersen et al.,PNAS (U.S.A.), 83:2642-2646 (1986); WT49 from Boss et al., PNAS(U.S.A.), 81:5199-5203 (1984); BURK from Karr et al., J. Immunol.,137:2886-2890 (1986); LG2 from Bull et al., PNAS (U.S.A.), 82:3405-3409(1985); DQBS4 and DQBS5 from Tsukamoto et al., Immunogenetics,25:343-346 (1987); AZH, BGE, and PGF from Lee et al., Immunogenetics,26:85-91 1987); and the related DXβ sequence was from Okada et al., PNAS(U.S.A.), 82:3410-3414 (1985). The PCR sequence illustrated for PGFmatched the published cDNA. The DQB alleles reported for the two IDDMpatients, DC and JR, were the only DQB3.1 alleles observed in 34 DR4patients.

FIGS. 5a, 5b, and 5c illustrate the alignment of HLA-DR-beta proteinsequences. DNA sequences (5b and 5c are continuation of 5a) of the DRβalleles were translated to the standard one-letter amino acid code(Table VIII, infra) and aligned to show patterns of homology. Theconventions used are the same as explained in the description for FIGS.4a and 4b, supra. See also Example I, infra.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Allelic sequence variations reported herein have been compared withconventional HLA classifications, and a new nomenclature based on thecorrespondence between current DQw serological specificities andsequence patterns is used. Specifically, the DNA-defined types DQB1,DQB2 and DQB3 designate sequences derived from the serologically definedDQw1, DQw2 and DQw3 haplotypes respectively, whereas DQB4 designatesthose sequences derived from DQ (blank) haplotypes, which are apparentlyhomogeneous. Employed in such nomenclature is the convention of usingGreek letters for the genetic loci and for the protein products (e.g.,the DQβ locus encoding the DQβ chain) and of using Roman capital lettersfollowed by a number to designate the specific allelic sequence variants(e.g., the DQB2 allele). Sequence variants which subdivide such typesare designated by a subtype number (e.g., DQB1.2 or DQA1.3).

However, the designation of DQα allelic variants do not alwayscorrespond to the DQw specificity; for example, the DQA4 type isassociated with both the DQw2 and DQw3 haplotypes. That is because theDQw2 and DQw3 specificities appear to be determined by polymorphicepitopes on the β-chain, independently of allelic variation on theα-chain.

"Posititively associated" with an autoimmune disease is a term usedherein to mean that the frequency of a marker is increased in patientswith the disease relative to controls (individuals without the disease).The converse meaning applies to the term "negatively associated" with anautoimmune disease, that is, the frequency of the marker is decreased inpatients relative to controls.

The terms "oligonucleotide" as used herein is defined as a moleculecomprised of two or more deoxyribonucleotides or ribonucleotides,preferably more than three. Its exact size will depend on many factors,which in turn depend on the ultimate function or use of theoligonucleotide. The oligonucleotide may be derived synthetically or bycloning.

The term "sequence-specific oligonucleotides" (SSOs) refers tooligonucleotides which will hybridize to one of the specific DNAsequences identified herein, which are regions of the loci where allelicvariations may occur. Such oligonucleotides have sequences spanning oneor more of the DNA regions being detected and are specific for one ormore of the regions being detected. One sequence-specificoligonucleotide is employed for each sequence to be detected, asdescribed further hereinbelow.

The term "monoclonal antibodies" as used herein refers to animmunoglobulin composition produced by a clonal population (or clone)derived through mitosis from a single antibody-producing cell. Unlessotherwise indicated, the term is not intended to be limited toantibodies of any particular mammalian species or isotype or toantibodies prepared in any given manner. The term is intended to includewhole antibody molecules as well as antigen-binding fragments (e.g.,Fab, F(ab')₂, Fc, Fc').

An "antibody-producing cell line" is a clonal population or clonederived through mitosis of a single antibody-producing cell capable ofstable growth in vitro for many generations. The term "cell line" refersto individual cells, harvested cells, and cultures containing cells solong as they are derived from cells of the cell line referred to.Preferably the cell lines remain viable for at least about six monthsand maintain the ability to produce the specified monoclonal antibodythrough at least about 50 passages.

As used herein, the term "incubation" means contacting antibodies andantigens under conditions (e.g., proper pH, temperature, time, medium,etc.) that allow for the formation of antigen/antibody complexes alsoreferred to as immune complexes. Also as used herein, "separating"refers to any method, usually washing, of separating a composition froma test support or immobilized antibody, such that any unbound antigen orantibody in the composition are removed and any immune complexes on thesupport remain intact. The selection of the appropriate incubation andseparation techniques is within the skill of the art.

HLA class II DR-beta genes have been isolated from HLA-typed IDDMpatients and HLA-matched controls and have been sequenced, resulting inregions of specific nucleotide and amino acid sequence which occur invarious combinations and which are associated with IDDM. These specificsequences can be used in DNA or protein diagnostic procedures todetermine genetic susceptibility to IDDM.

Four variant DR-beta sequences A-D found to be associated with IDDM areshown below. In each case, DNA sequences seen in the diabetic genomesproduce an alteration in one to three amino acid residues (underlined)of the DR-beta protein. The amino acids normally found in thesepositions are shown in parentheses. Sequences A-C are from the DRβIIIregion whereas sequence D is from the DRβI region. Sequence Dencompasses the "I-DE" (isoleucine, aspartic acid, glutamic acid atpositions 68, 71 and 72) epitope discussed infra.

A . . . GluLeu ArgLysSerGlu . . . GAGCTGCGTAAGTCTGAG . . .CTCGACGCATTCAGACTC . . . (Val,Ser,Leu,Pro,Asp,Ala)

B . . . GluGlu PheLeuArgPhe . . . GAGGAGTTCCTGCGCTTC . . .CTCCTCAAGGACGCGAAG . . . (Tyr,Asn,Ser,Asp)

C . . . Pro ValAlaGlu SerTrp . . . CCTGTCGCCGAGTCCTGG . . .CCGCAGCGGCTCAGGACC . . . (Asp,Ser) (Tyr)

D . . . AspIleLeuGluAspGluArg . . . GACATCCTGGAAGACGAGCGG . . .CTGTAGGACCTTCTGCTCGCC . . . (Leu,Phe) (Gln,Arg,Glu) (Lys,Arg,Ala)

Table I below shows the IDDM susceptibility and DR-beta variation withinthe DR3 and DR4 haplotypes. Table II shows the correlation between thehaplotypes and sequences A-D identified above. Sequences A, B and C arecorrelated with B8, DR3 vs. non B8, DR3 haplotypes.

                  TABLE I                                                         ______________________________________                                                    DR3        DR4                                                    ______________________________________                                        DRβ1     Not variable Variable (5)                                       DRβ3     Variable (2) Not variable                                       ______________________________________                                    

                  TABLE II                                                        ______________________________________                                                      Sequence                                                        Type      Gene      A     B       C   D                                       ______________________________________                                        DR4       beta-I    -     -       -   +                                       DR6       beta-I    -     -       -   +                                       DR6       beta-III  -     +       +   -                                       DR3       beta-III  +     +       +   -                                       DR3       beta-III  +     +       -   +                                       DR3       beta-III  -     +       -   -                                       ______________________________________                                    

Now abandoned U.S. Ser. No. 899,344 filed Aug. 22, 1986 entitled"Process for Detecting Specific Nucleotide Variations and GeneticPolymorphisms Present in Nucleic Acids," discloses the general method ofanalyzing allelic sequence variation by using allele-specificoligonucleotide (ASO) probes to hybridize to PCR amplified DNA in a dotblot format. That application, incorporated herein by reference and forwhich priority is claimed herein, lists some specific DNA and proteintranslation sequences derived from PCR cloning of several HLA class II(e.g., DRβ, DQα, and DQb) genes from a variety of HLA-typed individuals,either IDDM patients or controls. Some of such DNA sequences, identifiedby PCR and detectable by PCRot blot/ASO analysis, can function as usefulmarkers for disease susceptibility or differential diagnosis. One suchinformative set of DNA and translation sequences are the DQβ sequencesshown below in Tables III and IV (Tables IV and V in U.S. Ser. No.899,344), which respectively list the DNA and amino acid translationsequences for a number of allelic variants in the HLA-DQβ region. Thedesignations DR4, DR4' and DR4" therein are equivalent respectively tothe terms DQB3.2, DQB3.1 and DQB4 (blank).

                                      TABLE III                                   __________________________________________________________________________    HLA-DQβ (segment A):                                                      ##STR1##                                                                     HLA-DQβ (segments B and D):                                               ##STR2##                                                                     HLA-DQβ (segment C):                                                      ##STR3##                                                                     __________________________________________________________________________

    TABLE IV      - Alignment of HLA-DQβ      Protein Sequence      Exon-2:      ##STR4##     * = blank

Specifically, sequences for two variants of the serologically definedDR4 halotype listed as DR4 (DQB3.2) and DR4' (DQB3.1) in Tables III andIV are particularly informative. This DQβ subdivision of the DR4haplotype was correlated with a DQ8 RsaI RFLP in which one DR4 varianthad a RsaI fragment of 1.8 kb and another had a RsaI fragment of 1.5 kb(Arnheim et al., supra). Among DR4 individuals the 1.5 kb variant (DR4)was found to be positively associated with IDDM whereas the 1.8 kbfragment (DR4') was found to be negatively associated with IDDM asindicated by Table A below.

                  TABLE A                                                         ______________________________________                                                                     Relative                                                    IDDM     Controls Risk                                             ______________________________________                                        DR4          34/46      15/57    7.9                                          DR4 (1.5 kb; 32/46      10/57    10.8                                         DQB 3.2)                                                                      DR4'          2/46       5/57    0.47                                         (1.8 kb; DQB 3.1)                                                             ______________________________________                                         ##STR5##                                                                 

The DR4 (DQB3.2) and DR4' (DQB3.1) sequences differ by five nucleotidesubstitutions which result in three different amino acid changes. Two ofthe changes (Gly to Glu at codon 45 and Ala to Asp at codon 57) arenon-conservative and have major charge differences. The presence of avaline or alanine at codon 57 relative to aspartic acid is considered tobe positively associated with susceptibility to IDDM.

One of the most significant differences between the DQβ alleles seen inthe DR4 haplotypes, designated DQB3.1 and DQB3.2, is found at position57. (The numbering used herein corresponds to the amino acids in theprocessed protein.) The allele in the DQB3.1 haplotype, which is notassociated with IDDM susceptibility, has an aspartate residue atposition 57, whereas the allele in the DQB3.2 haplotype, which isstrongly associated with IDDM susceptibility, has an alanine residue atposition 57. Further, the DR3 haplotype which is positively associatedwith IDDM, has an alanine at position 57. The DR2 haplotype, negativelyassociated with IDDM, also has an aspartate residue at position 57.

Also, it was found that of two DQβ alleles in the DRw6 haplotypes, theallele positively associated with IDDM has a valine at position 57,whereas the allele negatively associated with IDDM has again anaspartate at position 57. Still further, it was found that the DR1haplotype which is moderately associated with IDDM has a valine atposition 57 in the DQβ locus.

Based on such observations, it was determined that the pattern of aminoacid variation at position 57 parallels the pattern of susceptibility toIDDM. Alleles containing alanine (hydrophobic residue) at position 57are most highly associated with IDDM, alleles containing valine(hydrophobic residue) at position 57 being moderately associated withIDDM, and alleles containing aspartate (charged residue) at 57 beingnegatively associated with IDDM.

A major exception to this pattern is the DQB2 allele of the DR7haplotype which has an alanine at position 57 as does the DR3 haplotypebut unlike DR3, is not associated with IDDM susceptibility.

Such a pattern extends to other genes also. For example, an allele ofthe DRβ-III gene within the DR3 haplotype, correlated with increasedIDDM susceptibility, has a valine at position 57, whereas most of theother DRβ alleles have aspartate at position 57. Thus, it was determinedthat the hydrophobic valine at position 57 of the DRβ-III allele isassociated with IDDM susceptibility as well as the hydrophobic alaninein the DQβ allele.

Table V below summarizes the sequences found around position 57 in anumber of genes. An asterisk in Table V indicates those haplotypesassociated with greatest IDDM susceptibility.

                  TABLE V                                                         ______________________________________                                        Variation in MHC class II β Proteins at Position 57                      ______________________________________                                        DQβ:            Position 57                                                                   ↓                                                        DR2, DR6.1    . . . GRPDAEY . . .                                             DR8           . . . - - L - - - - . . .                                       DR4.1 (aka DQw3.1)                                                                          . . . - P - - - - - . . .                                       DR4.2 (aka DQw3.2)                                                                          . . . - P - A - - - . . .                                                                   *                                                 DR3, DR7      . . . - L - A - - - . . .                                                                   *                                                 DR1, DR6.2    . . . - - - V - - - . . .                                                                   *                                          DRβ-III:                                                                        DR3.1, DR6.1, DR5                                                                           . . . GRPDAEY . . .                                             DR3.2, DR6.2, DR8                                                                           . . . - - - V - - S . . .                                                                   *                                          DRβ-I:                                                                          (consensus)   . . . GRPDAEY . . .                                             Dw15, DR8     . . . - - - S - - - . . .                                       DR7, DR9      . . . - - - V - - S . . .                                       DR6.2         . . . - - - A - DD . . .                                        DR5           . . . - - - - - E - - . . .                              I-Aβ:                                                                           d, k, b, u, s, q                                                                            . . . GRPDAEY . . .                                             f             . . . - - S - - - - . . .                                       NOD           . . . - - HS - - - . . .                                                                    * (IDDM -                                                                       suscep-                                                                       tible NOD                                                                     mouse)                                   I-Eβ:           Position 57                                                                   ↓                                                                      . . . GRPDAEN . . .                                      DPβ:                                                                                          . . . GRPAAEY . . .                                                           . . . - - - DE - - . . .                                                      . . . - - - - E - - . . .                                                     . . . - - - E - - - . . .                                                     . . . - - - DED - . . .                                  ______________________________________                                    

Table V indicates that three amino acid sequences encoded by DQβ allelesare associated With IDDM susceptibility. These sequences and thenucleotide sequences encoding them are listed in Table VI below. Alsolisted therein are the nucleotide and amino acid translation sequencesabout position 57 indicative of alleles negatively associated with IDDMsusceptibility. Codon 57 is underlined therein.

                  TABLE VI                                                        ______________________________________                                                                       IDDM                                           Alleles     Sequence           Association                                    ______________________________________                                        DQ-beta                                                                              (DR4):   Gly    Pro  Pro  Ala  Ala  ++                                        (DQw3.2) GGG    CCG  CCT   GCC GCC                                     DQ-beta                                                                              (DR3):   Gly    Leu  Pro  Ala  Ala  ++                                                 GGG    CTG  CCT   GCC GCC                                     DQ-beta                                                                              (DR1,6): Gly    Arg  Pro  Val  Ala  +                                                  GGG    CGG  CCT   GTT GCC                                     DQ-beta                                                                              (DR4):   Gly    Pro  Pro  Asp  Ala  -                                         (DQw3.1) GGG    CCG  CCT   GAC GCC                                     DQ-beta                                                                              (DR2):   Gly    Arg  Pro  Asp  Ala  -                                                  GGG    CGG  CCT   GAT GCC                                     ______________________________________                                    

The sequences in Table VI are considered to be marker DNA sequences fromHLA DQβ alleles that can be used to directly detect the identity of thecodon at position 57 that encodes for alanine, valine or aspartate.

Indirect ASO Analysis.

Although sequences around position 57 in the DQβ protein are those mostpositively associated with IDDM susceptibility, marker sequencesdirectly hybridizing thereto may not be optimal for inclusion inoligonucleotide probes. Since the DQβ alleles differ in other areas andare closely associated with other loci, such as DQα alleles, theidentity of the amino acid at position 57 of the DQβ alleles can bedetermined by the use of one or more oligonucleotide probes thathybridize to other regions of the DQβ, DQα or other HLA D regions.Further, such identification can be achieved by using one or more probeswherein one probe hybridizes to the region around position 57 andwherein one or more other probes hybridize to another HLA region inlinkage disequilibrium therewith.

For example, the DR3 haplotype is strongly associated with IDDMsusceptibility and contains an alanine at position 57 in DQβ. Althoughthis region can be detected directly with an ASO probe, such as GH74(see Summary, supra), the G-rich nature of that segment of DQβ leads toless than optimal probes because of the base mismatching potentialassociated therewith. The GH70 ASO probe (see Summary supra), however,is specific for a region about 90 bp upstream from codon 57 in an areathat is n6t as G/C-rich and thus avoids the base mismatching problem ofa direct probe for the position 57 segment. Thus, the binding of theGH70 ASO probe identifies indirectly the DQβ allele wherein codon 57encodes alanine.

Probes even farther away from the DQβ codon 57 position can also beused. For example, the GH66 ASO probe (see Summary supra) is specificfor the DR3 allele of the DQα locus, located about 12 kbp away from DQβ.However, as the DR3 allele of DQα locus has been shown to beconsistently linked with the DR3 allele of the DQβ locus, the binding ofthe GH66 probe identifies not only the DQα DR3 allele, but also the DQβDR3 allele. The use of the DQα locus also provides for discriminationbetween the IDDM susceptible DR3 haplotype and the less susceptible DR7haplotype.

Other Autoimmune Diseases

Susceptibility to other autoimmune diseases may also be related to codon57 polymorphism. The DRw6 susceptibility to Pemphigus vulgarisassociated with a rare DQβ allele (DQB1.3) which differs from thenon-susceptible alleles DQB1.2 and DQB1.1 only by a charge variation atposition 57 and is correlated with the Dw9 DRw6 subtype. Similarly, aDPβ allele found thus far only in celiac disease patients differs froman allele found in a homozygous typing cell (HTC) by an Ala-Aspsubstitution at position 57 (Bugawan et al., unpublished). [Celiacdisease is a digestive disorder characterized by a malabsorptionsyndrome affecting both children and adults precipitated by theingestion of gluten-containing foods; its etiology is unknown but ahereditary factor has been implicated.]

Described in Example III is the sequencing of the polymorphic secondexon of the DRβ1, DRβII and DQβ loci from three PV patients to discernany possible disease association with specific polymorphic class IIepitopes. In the DQβ loci, 3 of 4 DR4 haplotypes contained the DQB3.2allelic sequence variant present on 60-80% of control DR4 haplotypes,and one of the four DR4 haplotypes contained the DQB3.1 allele, presenton about 20-40% of control DR4 haplotypes [Erlich, et al., in Schacteret al. (eds): The Molecular Analysis of Histocompatibility Antigens, pp.93-109 (1987); Arnheim et al., PNAS (U.S.A.), (1985) 82:6970-6974; Kimet al., PNAS (U.S.A.), (1985) 82:8139-8143]. The two DR5 haplotypes alsocontained the DQB3.1 allelic sequence variant present on all control DR5haplotypes. Thus, the distribution of DQβ alleles was essentially thesame in patients, and in DR-matched controls. In this small sample, allthree patients were DQB3.1/DQB3.2 heterozygotes.

In the DRβI locus, however, a potentially interesting pattern could bediscerned. All three PV patients contained a DR4 haplotype with a DRβIallelic sequence variant associated with the MLC-defined subtype, Dw10.In the U.S. population the frequency of the Dw10 subtype among DR4haplotypes is estimated to be approximately 10% [Hansen et al., Brit.Med. Bull. (1987) 43:203-216]. This observation was confirmed usingoligonucleotide probes rather than sequence analysis with virtually 100%of DR4 PV patients containing the Dw10 epitope. Since this epitope isassociated with the MLC-defined type, Dw10, it is likely to berecognized by the T-cell receptor. These results suggest that for DR4associated susceptibility the amino acid residues isoleucine, asparticacid and glutamic acid at positions 68, 71, and 72 of the DR4 DRβI chainplay a role in PV autoimmunity. Such residues define an epitope hereinidentified as the "I-DE" epitope. [See Example III.]

As indicated in Example IV, the same "I-DE" epitope around codon 70 isalso present on a subset of DRw6 haplotypes, but said epitope was notshown to be positively associated with PV in such DRw6 haplotypes. Asthe "I-DE" shared epitope cannot account for the DRw6 susceptibility,further research was performed to find sequences conferring PVsusceptibility within the DRw6 haplotype by determining the sequences ofthe DQβ alleles of two DR5/DRw6 PV patients by methods exemplified inExample III. Both patient DRw6 haplotypes were found to contain apreviously unknown DQβ allele, which was designated DQB1.3 (see HU129sequence of FIG. 4). The DQB1.3 allele differs from the DR1 DQβ allele,DQB1.1 by only a valine to aspartic acid substitution at position 57.Analogously, it differs from the rare DR2 AZH DQβ allele, DQB1.2, byonly a serine to aspartic acid substitution at position 57. At thenucleotide level, the DQB1.3 allele is identical in the region aroundposition 57 to the DR2 Dw12 (DQB1.5) allele, DQβ, and to the most commonDRw6 DQβ allele DQB1.6.

To determine the frequency of the DQB1.3 allele among PV patient andcontrol DRw6 haplotypes, pairs of sequence-specific oligonucleotide(SSO) probes were used to identify both the DR1-like DQβ frameworksequence and the sequence around codon 57. Exemplary SSO probes are asfollows:

(1) GH69 a 21-mer which identifies a DR1-like DQβ framework and has thenucleotide sequence GATGTGTCTGGTCACACCCCG;

(2) GH80 a 19-mer which identifies a DRw6 like framework and has thenucleoitide sequence TCTTGTAACCAGACACATC;

(3) CRX03 a 19-mer which identifies a sequence about codon 57 whereincodon 57 encodes for aspartic acid and has the nucleotide sequenceTCGGCGTCAGGCCGCCCCT; and

(4) CRX02 a 19-mer which identifies a sequence around codon 57 whereincodon 57 encodes for valine and has the nucleotide sequenceTCGGCAACAGGCCGCCCCT.

Using the above-designated probes, a pattern of hybridization can beused to identify and distinguish specific alleles. The following chartexemplifies the use of such a method wherein a plus sign (+) indicateshybridization of the SSO probe to the target DNA sample.

    ______________________________________                                                 Probes                                                               Alleles    GH69    GH80        CRX03 CRX02                                    ______________________________________                                        Dw9     1.3    +                 +                                                    1.1    +                       +                                      Dw18    1.6            +         +                                            Dw19    1.7            +               +                                      ______________________________________                                    

Using this approach, 11 of the 13 DRw6 patient haplotypes (85%)contained the DQB1.3 allele, whereas only one of the 13 control DRw6haplotypes (8%) contained said allele. [The other two DRw6 patients hadDQβ alleles that had the DR1-like framework (identified by GH69) butfailed to hybridize with either the CRX03 or the CRX02 probes.]

The findings indicate that the DRw6 associated PV susceptibility couldbe conferred by the rare DQβ allele DQB1.3 that differs from the commonDQδ allele DQB1.1 by only one residue. Such a single charge differenceof the polymorphic residue at position 57 of the DQ beta chainassociated with the DRw6 associated PV susceptibility correlates withthat found for the DR4 and DR3 associated susceptibility for IDDM.However, in the case of PV, it is clear that it is the allele ratherthan the epitope around position 57 that confers susceptibility becausethe most common DRw6 DQβ allele that is, DQB1.6, which is not associatedwith PV, has the same sequence around position 57. Since the DQB1.6allele differs at other regions of its sequence (see FIG. 4), it ispossible to differentiate the DQB1.6 common allele from the rare DQB1.3allele by the use of pairs of SSO probes, for example, as indicatedimmediately above, the GH69, GH80, CRX03, and CRX02 probes or onessubstantially similar thereto.

The conclusion that the novel DQβ allele accounts for the DRw6associated susceptibility to PV is consistent with DQβ RFLP analysis ofSfazer et al., supra (1987).

Viral Mimicry and Autoimmune Disease

Although the pattern of DQβ allelic variation clearly implicatesposition 57 in autoimmune predisposition, this region does not appear tobe the only class II epitope within susceptibility conferring haplotypeswhich contribute to autoimmune disease. No class II sequences have beenfound to be uniquely associated with IDDM. That observation suggeststhat "normal" class II alleles confer susceptibility, or that thesusceptibility genes reside elsewhere in the MHC. Given the estimates ofpenetrance and concordance (50% for monozygetic twins and 25% for HLAidentical sibs) for IDDM [Henson et al., Mol. Biol. Med. (1986),3:129-136], it is not surprising that some unaffected individualscontain putative class II susceptibility genes.

It appears that some environmental "triggering" agent, such as viralinfection, is required for the disease to develop in susceptibleindividuals. The homology between DQβ alleles and rubella, a virusimplicated in IDDM pathogenesis suggests a viral triggering mechanism.

Viruses have evolved mechanisms to evade their hosts immune defenses(McChesney et al., supra; Srinivasappa et al., supra), and some of thesemechanisms appear to involve mimicry of vital MHC epitope. The presentinvention relates to the identification of regions of homology betweenthe HLA-DQ proteins and human viral pathogens. [See Example VI.] TableVII summarizes the major homologies observed to the Epstein-Barr virus(EBV), the genome for which has been completely sequenced. [Baer et al,Nature, 310:207-211.]

                  TABLE VII                                                       ______________________________________                                        Homologies Between HLA-DQβ Alleles and EBV                               DQβ                                                                            HLA       EBV                  ORF  ORF                                 allele                                                                              epitope   homology pos.   phase                                                                              size name                                ______________________________________                                        DQB1.4                                                                              GRPDAEY   RPDAE    167112 R3   101  BNLF26                              (DR2)                                                                         DQB2  GLPAAEY   GLPAA    792    R1   134                                      (DR3)                    67134  R3   188                                                               80004  R1   30                                                                118884 R3   57                                                                134242 F1   72                                                       PAAEY    73713  R3   1374 BOLF1                               DQB3.2                                                                              GPPAAEY   GPPAA    12404* F1   129  BWRF1                               (DR4)                    61311  R1   20                                                                100137**                                                                             F3   872  BERF4                                                        100257 F3   872  BERF4                                               PPAAEY   73713  R3   1374 BOLF1                               ______________________________________                                         *(repeated 12 times as part of the 3072 bp `IR1` repeat)                      **(directly repeated 6 times)                                            

Only one homology was seen between the peptide and potential epitopecentered at position 57 in the DQB1.4 (DR2) allele and the entire EBVgenome. However, six matches were seen to the peptide from the DRB2(DR3) allele, and 21 were seen to the GPPAA peptide from the DQB3.2(DR4) allele. Many of the later homologies were found in repeatedsegments of the EBV genome, including one segment of five amino acidsdirectly repeated six times. In addition, the E1 envelope protein ofrubella [Nakhasi et al., J. Biol. Chem., (1986), 261:16616-16621], avirus implicated in IDDM pathogenesis [Rubenstein et al., Diabetes,(1982), 31:1088-1091], contains the GPPAA peptide at position 261.Exposure of a fetus to the rubella virus leads to a congenitalinfection, and a high risk for diabetes. Based on the homology betweenviral pathogens (such as EBV and rubella) and the DQβ peptide Gly ProPro Ala Glu, such region could also serve as a target for molecularmimicry [Oldstone et al., supra] with an immune response to an infectingvirus possibly leading to an attack on self cells.

Further, viruses other than EBV and rubella have been implicated withIDDM such as Coxsackie and cytomegalovirus (CMV) (Yoon et al., supra).

The viruses are likely mimicking HLA class II genes, and in particularthe HLA DQβ genes around codon 57, delaying or modifying the onset of aneffective immune response. If the immune system actually responds to themimicked HLA epitope, then normal regulation of the immune system couldbe perturbed, possible leading to autoimmune disease.

Autoimmune disease could result from a series of factors: 1) inheritanceof HLA alleles which are being mimicked by viruses, 2) infection by avirus mimicking the host's HLA class II alleles, 3) an immune responseby the host to the mimicked epitopes, 4) perturbation of immuneregulation of autoimmune responses, 5) development of an autoimmuneresponse, and 6) progressive tissue destruction leading to an autoimmunedisease.

An example of this mechanism could involve a person inheriting theDQB3.2 allele correlating with increased risk to IDDM followed byinfection by the rubella virus, which is also correlated with IDDM, andwhich contains an epitope in its E1 envelope protein which specificallymimics a portion of the DQB3.2 protein. After infection by the virus,the person may elicit an immune response involving antibodies or T cellsdirected against the E1 protein, and cross-reactive to the DQB3.2protein. This antibody or T cell response interferes with the normalfunction of the HLA DQ protein, leading to an autoimmune response andIDDM. The target of the final autoimmunity may be determined by thelocation of the viral infection, in this case the beta cells of theislets of Langerhans. Increased degrees of risk could be ascertained asan individual is shown to have the DQB3.2 allele, followed by infectionby the rubella virus, followed by the appearance of anti-rubellaantibodies cross-reactive to the HLA DQ-beta protein.

Diagnostic tests to determine whether an individual with allelesassociated with an autoimmune susceptibility has been infected by avirus correspondingly associated with susceptibility to an autoimmunedisease (based on the virus having homology to segments of the HLA classII alleles) could be used to determine an effective prophylactic ortherapeutic treatment plan for such individual. For example, vaccines,immunotoxins, immuno-antigens, peptides corresponding to the epitope ofthe mimicked region, or anti-idiotype antibodies could then be used toprevent or reverse an immune response against epitopes presented by thepathogenic organisms.

An example of such a diagnostic test could be detecting an immuneresponse (i.e., antibody production or reactive immune cells) asreported in Schwinnbeck et al., J. Exp. Med., 166:173-181 (1987), whereantibodies against a portion of the class I HLA molecule B27 aredetected by attaching a synthetic peptide to a solid support, treatingit with a dilution of the test serum, washing the solid support, andthen testing whether any antibodies are retained. Other examples of sucha diagnostic test could be either by directly probing for the viral DNAgenome, as is done with the HIV (AIDS) virus, in Kwok et al., J. Virol.,61:1690-1694 (1987), or by indirectly assaying for antibodies arisingfrom infection, as in agglutination tests for CMV antibodies.

Dw4

The DR4, Dw10 DRβI allele is associated with susceptibility to both PVand to IDDM. That allele contains the nucleotide sequence which encodesthe I-DE amino acid sequence in the third hypervariable region (HV3;segment D in FIG. 5) around positions 68-72 of the DRβI chain. AnotherDR4 DRβI allele, Dw4, also polymorphic in the same hypervariable region(HV3; segment D in FIG. 5), has the sequence GGAGCAGAAGCGGGCCGCG aroundpositions 68-72, and is also associated with IDDM. Thus, most DR4+ IDDMpatients are either DR4, Dw4 or DR4, Dw10, indicating that both the DQβallelic variants (see above) and the DRβI allelic variants contribute toautoimmunity. The DW4 variant can be distinguished from the other DR4,DRβI alleles by sequence specific oligonucleotide (SSO) analysis.

The DR4 haplotype, Dw4 subtype is also associated with rheumatoidarthritis (RA), as is DR4, Dw14. [Roudier et al., Abstract from AmericanRheumatism Association (Western Region) Meeting in San Diego, Calif.,Nov. 5-7, 1987, page 15.] A hexapeptide from the HV3 region of the DRβIchain at amino acids 69-74, that distinguishes Dw4 from the other DR4,DRβI alleles is shared by the Epstein-Barr virus (EBV) open readingframe BALF4 and may serve as a target for molecular mimicry.

Detection Means

The above-mentioned DNA sequences may be detected by DNA hybridizationprobe technology. In one example, which is not exclusive, the samplesuspected of containing the genetic marker is spotted directly on aseries of membranes and each membrane is hybridized with a differentlabeled oligonucleotide probe that is specific for the particularsequence variation. One procedure for spotting the sample on a membraneis described by Kafotos et al., Nucleic Acids Research, 7:1541-1552(1979).

Briefly, the DNA sample affixed to the membrane may be pretreated with aprehybridization solution containing sodium dodecyl sulfate, Ficoll,serum album in and various salts prior to the probe being added. Then, alabeled oligonucleotide probe that is specific to each sequence to bedetected is added to a hybridization solution similar to theprehybridization solution. The hybridization solution is applied to themembrane and the membrane is subjected to hybridization conditions thatwill depend on the probe type and length, type and concentration ofingredients, etc. Generally, hybridization is carried out at about25-75° C., preferably 35° to 65° C., for 0.25-50 hours, preferably lessthan three hours. The greater the stringency of conditions, the greaterthe required complementarity for hybridization between the probe andsample. If the background level is high, stringency may be increasedaccordingly. The stringency can also be incorporated in the wash.

After the hybridization the sample is washed of unhybridized probe usingany suitable means such as by washing one or more times with varyingconcentrations of standard saline phosphate EDTA (SSPE) (180 mM NaCl, 10mM Na₂ HPO₄ and 1M EDTA, pH 7.4) solutions at 25°-75° C. for about 10minutes to one hour, depending on the temperature. The label is thendetected by using any appropriate detection techniques.

The sequence-specific oligonucleotide that may be employed herein is anoligonucleotide that may be prepared using any suitable method, such as,for example, the organic synthesis of a nucleic acid from nucleosidederivatives. This synthesis may be performed in solution or on a solidsupport. One type of organic synthesis is the phosphotriester method,which has been utilized to prepare gene fragments or short genes. In thephosphotriester method, oligonucleotides are prepared that can then bejoined together to form longer nucleic acids. For a description of thismethod, see Narang, S. A., et al., Meth. Enzymol., 68, 90 (1979) andU.S. Pat. No. 4,356,270. The patent describes the synthesis and cloningof the somatostatin gene.

A second type of organic synthesis is the phosphodiester method, whichhas been utilized to prepare tRNA gene. See Brown, E. L., et al., Meth.Enzymol., 68, 109 (1979) for a description of this method. As in thephosphotriester method, the phosphodiester method involves synthesis ofoligonucleotides that are subsequently joined together to form thedesired nucleic acid.

Automated embodiments of these methods may also be employed. In one suchautomated embodiment diethylphosphoramidites are used as startingmaterials and may be synthesized as described by Beaucage et al.,Tetrahedron Letters, 22:1859-1862 (1981). One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066. It is also possible to use a primer which has beenisolated from a biological source (such as a restriction endonucleasedigest).

The sequence-specific oligonucleotide must encompass the region of thesequence which spans the nucleotide variation being detected and must bespecific for the nucleotide variation being detected. For example, fouroligonucleotides may be prepared, each of which contains the nucleotidesequence site characteristic of each of the four DNA sequences herein.Each oligonucleotide would be hybridized to duplicates of the samesample to determine whether the sample contains one or more of theregions of the locus where allelic variations may occur which arecharacteristic of IDDM or PV.

The length of the sequence-specific oligonucleotide will depend on manyfactors, including the source of oligonucleotide and the nucleotidecomposition. For purposes herein, the oligonucleotide typically contains15-25 nucleotides, although it may contain more or fewer nucleotides.While oligonucleotides which are at least 19-mers in length may enhancespecificity and/or sensitivity, probes which are less than 19-mers,e.g., 16-mers, show more sequence-specific discrimination, presumablybecause a single mismatch is more destabilizing. If amplification of thesample is carried out as described below prior to detection with theprobe, amplification increases specificity so that a longer probe lengthis less critical, and hybridization and washing temperatures can belowered for the same salt concentration. Therefore, in such as case itis preferred to use probes which are less than 19-mers.

Where the sample is first placed on the membrane and then detected withthe oligonucleotide, the oligonucleotide must be labeled with a suitablelabel moiety, which may be detected by spectroscopic, photochemical,biochemical, immunochemical or chemical means. Immunochemical meansinclude antibodies which are capable of forming a complex with theoligonucleotide under suitable conditions, and biochemical means includepolypeptides or lectins capable of forming a complex with theoligonucleotide under the appropriate conditions. Examples includefluorescent dyes, electron-dense reagents, enzymes capable of depositinginsoluble reaction products or being detected chronogenically, such asalkaline phosphatase, a radioactive label such as ³² P, or biotin. Ifbiotin is employed, a spacer arm may be utilized to attach it to theoligonucleotide. Preferably, the labels used are non-radioactive.

In a "reverse" dot blot format, a labeled sequence-specificoligonucleotide probe capable of hybridizing with one of the DNAsequences is spotted on (affixed to) the membrane under prehybridizationconditions as described above. The sample is then added to thepretreated membrane under hybridization conditions as described above.Then the labeled oligonucleotide or a fragment thereof is released fromthe membrane in such a way that a detection means can be used todetermine if a sequence in the sample hybridized to the labeledoligonucleotide. The release may take place, for example, by adding arestriction enzyme to the membrane which recognizes a restriction sitein the probe. This procedure, known as oligomer restriction, isdescribed more fully in EP Patent Publication 164,054 published Dec. 11,1985, the disclosure of which is incorporated herein by reference.

Alternatively, a sequence specific oligonucleotide immobilized to themembrane could bind or "capture" a target DNA strand (PCR-amplified).This "captured" strand could be detected by a second lableed probe. Thesecond oligonucleotide probe could be either locus-specific orallele-specific.

In an alternative method for detecting the DNA sequences herein, thesample to be analyzed is first amplified using DNA polymerase, fournucleotide triphosphates and two primers. Briefly, this amplificationprocess involves the steps of:

(a) treating a DNA sample suspected of containing one or more of thefour IDDM genetic marker sequences, together or sequentially, with fourdifferent nucleotide triphosphates, an agent for polymerization of thenucleotide triphosphates, and one deoxyribonucleotide primer for eachstrand of each DNA suspected of containing the IDDM or PV geneticmarkers under hybridizing conditions, such that for each DNA strandcontaining each different genetic marker to be detected, an extensionproduct of each primer is synthesized which is complementary to each DNAstrand, wherein said primer(s) are selected so as to be substantiallycomplementary to each DNA strand containing each different geneticmarker, such that the extension product synthesized from one primer,when it is separated from its complement, can serve as a template forsynthesis of the extension product of the other primer;

(b) treating the sample under denaturing conditions to separate theprimer extension products from their templates if the sequence(s) to bedetected are present; and

(c) treating the sample, together or sequentially, with said fournucleotide triphosphates, an agent for polymerization of the nucleotidetriphosphates, and oligonucleotide primers such that a primer extensionproduct is synthesized using each of the single strands produced in step(b) as a template, wherein steps (b) and (c) are repeated a sufficientnumber of times to result in detectable amplification of the nucleicacid containing the sequence(s) if present.

The sample is then affixed to a membrane and detected with asequence-specific probe as described above. Preferably, steps (b) and(c) are repeated at least five times, and more preferably 15-30 times ifthe sample contains human genomic DNA. If the sample comprises cells,preferably they are heated before step (a) to expose the DNA therein tothe reagents. This step avoids extraction of the DNA prior to reagentaddition.

In a "reverse" dot blot format, at least one of the primers and/or atleast one of the four nucleotide triphosphates used in the amplificationchain reaction is labeled with a detectable label, so that the resultingamplified sequence is labeled. These labeled moieties may be presentinitially in the reaction mixture or added during a later cycle. Then anunlabeled sequence-specific oligonucleotide capable of hybridizing withthe amplified sequence(s), if the sequence(s) is/are present, is spottedon (affixed to) the membrane under prehybridization conditions asdescribed above. The amplified sample is then added to the pretreatedmembrane under hybridization conditions as described above. Finally,detection means are used to determine if an amplified sequence in theDNA sample has hybridized to the oligonucleotide affixed to themembrane. Hybridization will occur only if the membrane-bound sequencecontaining the variation is present in the amplification product.

As indicated above, variations of this method include use of anunlabeled PCR target, an unlabeled immobilized allele-specific probe anda labeled oligonucleotide probe in a sandwich assay.

The amplification method provides for improved specificity andsensitivity of the probe; an interpretable signal can be obtained with a0.04 μg sample in six hours. Also, if the amount of sample spotted on amembrane is increased to 0.1-0.5 μg, non-isotopically labeledoligonucleotides may be utilized in the amplification process ratherthan the radioactive probes used in previous methods. Finally, asmentioned above, the amplification process is applicable to use ofsequence-specific oligonucleotides less than 19-mers in size, thusallowing use of more discriminatory sequence-specific oligonucleotides.

In a variation of the amplification procedure, a thermostable enzyme,such as one purified from Thermus aquaticus, may be utilized as the DNApolymerase in a temperature-cycled chain reaction. The thermostableenzyme refers to an enzyme which is stable to heat and is heat resistantand catalyzes (facilitates) combination of the nucleotides in the propermanner to form the primer extension products that are complementary toeach DNA strand.

In this latter variation of the technique, the primers and nucleotidetriphosphates are added to the sample, the mixture is heated and thencooled, and then the enzyme is added, the mixture is then heated toabout 90°-100° C. to denature the DNA and then cooled to about 35°-40°C., and the cycles are repeated until the desired amount ofamplification takes place. This process may also be automated. Theamplification process using the thermostable enzyme is described orefully in copending U.S. application Ser. Nos. 899,513 and 063,647 filedrespectively Aug. 22, 1986 and Jun. 17, 1987, entitled "Process forAmplifying, Detecting, and/or Cloning Nucleic Acid Sequences Using aThermostable Enzyme," the disclosures of which are incorporated hereinby reference.

The invention herein also contemplates a kit format which comprises apackaged multicontainer unit having containers for each labeledsequence-specific DNA probe. The kit may optionally contain a means todetect the label (such as an avidin-enzyme conjugate and enzymesubstrate and chromogen if the label is biotin). In addition, the kitmay include a container that has a positive control for the probecontaining one or more DNA strands with the sequence to be detected anda negative control for the probe that does not contain the DNA strandshaving any of the sequences to be detected.

One method for detecting the amino acid sequences in a protein samplethat are associated with IDDM or PV involves the use of an immunoassayemploying one or more antibodies that bind to one or more of the fouramino acid sequences. While the antibodies may be polyclonal ormonoclonal, monoclonal antibodies are preferred in view of theirspecificity and affinity for the antigen.

Polyclonal antibodies may be prepared by well-known methods whichinvolve synthesizing a peptide containing one or more of the amino acidsequences associated with IDDM or PV, purifying the peptide, attaching acarrier protein to the peptide by standard techniques, and injecting ahost such as a rabbit, rat, goat, mouse, etc. with the peptide. The seraare extracted from the host by known methods and screened to obtainpolyclonal antibodies which are specific to the peptide immunogen. Thepeptide may be synthesized by the solid phase synthesis method describedby Merrifield, R. B., Adv. Enzymol. Relat. Areas Mol. Biol., 32:221-296(1969) and in "The Chemistry of Polypeptides" (P. G. Katsoyannis, ed.),pp. 336-361, Plenum, N.Y. (1973), the disclosures of which areincorporated herein by reference. The peptide is then purified and maybe conjugated to keyhold limpet hemocyanin (KLH) or bovine serum albumin(BSA). This may be accomplished via a sulfhydryl group, if the peptidecontains a cysteine residue, using a heterobifunctional crosslinkingreagent such as N-maleimido-6-amino caproyl ester of1-hydroxy-2-nitrobenzene-4-sulfonic acid sodium salt.

The monoclonal antibody will normally be of rodent or human originbecause of the availability of murine, rat, and human tumor cell linesthat may be used to produce immortal hybrid cell lines that secretemonoclonal antibody. The antibody may be of any isotype, but ispreferably an IgG, IgM or IgA, most preferably an IgG2a.

The murine monoclonal antibodies may be produced by immunizing the hostwith the peptide mentioned above. The host may be inoculatedintraperitoneally with an immunogenic amount of the peptide and thenboosted with similar amounts of the immunogenic peptide. Spleens orlymphoid tissue is collected from the immunized mice a few days afterthe final boost and a cell suspension is prepared therefrom for use inthe fusion.

Hybridomas may be prepared from the splenocytes or lymphoid tissue and atumor (myeloma) partner using the general somatic cell hybridizationtechnique of Koehler, B. and Milstein, C., Nature, 256:495-497 (1975)and of Koehler, B. et al., Eur. J. Immunol., 6:511-519 (1976). Preferredmyeloma cells for this purpose are those which fuse efficiently, supportstable, high-level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred myeloma cell lines are murine myelomalines, such as those derived from MOPC-21 and MOPC-11 mouse tumorsavailable from the Salk Institute, Cell Distribution Center, San Diego,Calif., U.S.A., or P3X63-Ag8.653 (653) and Sp2/0-Ag14 (SP2/0) myelomalines available from the American Type Culture Collection, Rockville,Md., U.S.A., under ATCC CRL Nos. 1580 and 1581, respectively.

Basically, the technique involves fusing the appropriate tumor cells andsplenocytes or lymphoid tissue using a fusogen such as polyethyleneglycol. After the fusion the cells are separated from the fusion mediumand grown on a selective growth medium, such as HAT medium, to eliminateunhybridized parent cells and to select only those hybridomas that areresistant to the medium and immortal. The hybridomas may be expanded, ifdesired, and supernatants may be assayed by conventional immunoassayprocedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescenceimmunoassay) using the immunizing agent as antigen. Positive clones maybe characterized further to determine whether they meet the criteria ofthe antibodies of the invention. For example, the antigen-bindingability of the antibodies may be evaluated in vitro by immunoblots,ELISAs and antigen neutralizing tests.

A preferred procedure for making a hybrid cell line that secretes humanantibodies against the amino acid genetic markers is somatic cellhybridization using a mouse x human parent hybrid cell line and a humancell line producing sufficiently high levels of such antibodies. Thehuman cell line may be obtained from volunteers immunized with thepeptide(s) described above. The human cell line may be transformed withEpstein-Barr virus (EBV) as described, for example, by Foung, et al., J.Immunol. Methods, 70:83-90 (1984).

When EBV transformation is employed, the most successful approaches havebeen either to pre-select the population of B cells to be transformed orto post-select the antigen-specific transformed populations by panningor rosetting techniques, as described by Kozbar, et al., Scan. J.Immunol., 10:187-194 (1979) and Steinitz, et al., J. Clin. Lab. Immun.,2:1-7 (1979). Recently EBV transformation has been combined with cellfusion to generate human monoclonal antibodies (see, e.g., Foung et al.,J. Immun. Meth., 70:83-90 (1984)), due to instability of immunoglobulinsecretion by hybridomas when compared to EBV lymphoblastoid cell lines,and higher frequencies of rescue of the antigen-specific populations.EBV most frequently infects and transforms IgM-bearing B cells, but Bcells secreting other classes of Ig can also be made into long-termlines using the EBV fusion technique, as described by Brown and Miller,J. Immunol., 128:24-29 (1982).

The cell lines which produce the monoclonal antibodies may be grown invitro in suitable culture medium such as Iscove's medium, Dulbecco'sModified Eagle's Medium, or RPMI-1640 medium from Gibco, Grand Island,N.Y., or in vivo in syngeneic or immunodeficient laboratory animals. Ifdesired, the antibody may be separated from the culture medium or bodyfluid, as the case may be, by conventional techniques such as ammoniumsulfate precipitation, hydroxyapatite chromatography, ion exchangechromatography, affinity chromatography, electrophoresis,microfiltration, and ultracentrifugation.

The antibodies herein may be used to detect the presence or absence ofone or more of the four amino acid sequences associated with IDDM inwhite blood cells expressing the HLA class II antigens. The cells may beincubated in the presence of the antibody, and the presence or absenceand/or degree of reaction (antibody-peptide binding) can be determinedby any of a variety of methods used to determine or quantitateantibody/antigen interactions (e.g., fluorescence, enzyme-linkedimmunoassay (ELISA), and cell killing using antibody and complement bystandard methods). The antibody employed is preferably a monoclonalantibody.

For use in solid phase immunoassays, the antibodies employed in thepresent invention can be immobilized on any appropriate solid testsupport by any appropriate technique. The solid test support can be anysuitable insoluble carrier material for the binding of antibodies inimmunoassays. Many such materials are known in the art, including, butnot limited to, nitrocellulose sheets or filters; agarose, resin,plastic (e.g., PVC or polystyrene) latex, or metal beads; plasticvessels; and the like. Many methods of immobilizing antibodies are alsoknown in the art. See, e.g., Silman et al., Ann. Rev. Biochem., 35:873(1966); Melrose, Rev. Pure & App. Chem., 21:83 (1971); Cuatrecafas, etal., Meth. Enzym., Vol. 22 (1971). Such methods include covalentcoupling, direct adsorption, physical entrapment, and attachment to aprotein-coated surface. In the latter method, the surface is firstcoated with a water-insoluble protein such as zein, collagen,fibrinogen, keratin, glutelin, etc. The antibody is attached by simplycontacting the protein-coated surface with an aqueous solution of theantibody and allowing it to dry.

Any combination of support and binding technique which leaves theantibody immunoreactive, yet sufficiently immobilizes the antibody sothat it can be retained with any bound antigen during a washing, can beemployed in the present invention. A preferred solid test support is aplastic bead.

In the sandwich immunoassay, a labeled antibody is employed to measurethe amount of antigen bound by the immobilized monoclonal antibody. Thelabel can be any type that allows for the detection of the antibody whenbound to a support. Generally, the label directly or indirectly resultsin a signal which is measurable and related to the amount of labelpresent in the sample. For example, directly measurable labels caninclude radiolabels (e.g., ¹²⁵ I, ³⁵ S, ¹⁴ C, etc.). A preferreddirectly measurable label is an enzyme, conjugated to the antibody,which produces a color reaction in the presence of the appropriatesubstrate (e.g. horseradish peroxidase/o-phenylenediamine). An exampleof an indirectly measurable label would be anti body that has beenbiotinylated. The presence of this label is measured by contacting itwith a solution containing a labeled avidin complex, whereby the avidinbecomes bound to the biotinylated antibody. The label associated withthe avidin is then measured. A preferred example of an indirect label isthe avidin/biotin system employing an enzyme conjugated to the avidin,the enzyme producing a color reaction as described above. It is to beunderstood, however, that the term "label" is used in its broadest senseand can include, for example, employing "labeled" antibodies where thelabel is a xenotypic or isotypic difference from the immobilizedantibody, so that the presence of "labeled" antibodies is detectable byincubation with an anti-xenotypic or anti-isotypic antibody carrying adirectly detectable label.

Whatever label is selected, it results in a signal which can be measuredand is related to the amount of label in a sample. Common signals areradiation levels (when radioisotopes are used), optical density (e.g.,when enzyme color reactions are used), and fluorescence (whenfluorescent compounds are used). It is preferred to employ anonradioactive signal, such as optical density (or color intensity)produced by an enzyme reaction. Numerous enzyme/substrate combinationsare known in the immunoassay art which can produce a suitable signal.See, e.g., U.S. Pat. Nos. 4,323,647 and 4,190,496, the disclosures ofwhich are incorporated herein.

For diagnostic use, the antibodies will typically be distributed inmulticontainer kit form. These kits will typically contain theantibody(ies) in labeled or unlabeled form in suitable containers, anydetectable ligand reactive with unlabeled antibody if it is used,reagents for the incubations and washings if necessary, reagents fordetecting the label moiety to be detected, such as substrates orderivatizing agents depending on the nature of the label, productinserts and instructions, and a positive control associated with IDDM orPV, such as a cell containing the HLA class II antigens associated withIDDM or PV. The antibodies in the kit may be affinity purified if theyare polyclonal.

The following examples illustrate various embodiments of the inventionand are not intended to be limiting in any respect. In the examples allparts and percentages are by weight if solid and by volume if liquid,and all temperatures are in degrees Centigrade, unless otherwiseindicated.

EXAMPLE I

This example illustrates how four DR-β sequences associated with IDDMwere identified.

I. Analysis of HLA-DR-β Sequences

Several HLA class II beta genes were isolated from clinical bloodsamples of diverse HLA-typed IDDM individuals (from University ofPittsburgh clinic and from cell lines from IDDM patients available fromthe Human Genetic Mutant Cell Repository, Camden, N.J.) and non-diabeticcontrols (homozygous typing cells) using cloning methods. In one suchmethod, which is a standard method, human genomic DNA was isolated fromthe patient samples using essentially the method of Maniatis et al.,Molecular Cloning: A Laboratory Manual (1982), 280-281 or prepared fromthe buffy coat fraction, which is composed primarily of peripheral bloodlymphocytes, as described by Saiki et al., Bio/Technology, 3:1008-1012(1985). This DNA was then cloned as full genomic libraries intobacteriophage vectors, as described in Maniatis, supra, pp. 269-294.Individual clones for the HLA-DRβ genes were selected by hybridizationto radioactive cDNA probes (Maniatis et al., pp. 309-328) andcharacterized by restriction mapping. See U.S. Pat. No. 4,582,788 issuedApr. 15, 1986. Individual clones from IDDM patients were assigned toDR-typed haplotypes by comparing the clone restriction map with the RFLPsegregation pattern within the patients' family. Finally, smallfragments of these clones representing the variable second exon weresubcloned (Maniatis, pp. 390-402) into the M13mp10 cloning vector, whichis publicly available from Boehringer-Mannheim.

In an alternative procedure for cloning the genes, amplification of therelevant portion (the second exon) of the gene was carried out asdescribed below.

A total of 1 microgram of each isolated human genomic DNA was amplifiedin an initial 100 μl reaction volume containing 10 μl of a solutioncontaining 100 mM Tris.HCl buffer (pH 7.5), 500 mM NaCl, and 100 mMMgCl₂, 10 μl of 10 μM of primer GH46, 10 μl of 10 μM of primer GH50, 15μl of 40 mM dNTP (contains 10 mM each of dATP, dCTP, dGTP and TTP), and45 μl of water. Primers GH46 and GH50 have the following sequences:

5'-CCGGATCCTTCGTGTCCCCACAGCACG-3' (GH46)

5'-CTCCCCAACCCCGTAGTTGTGTCTGCA-3' (GH50)

These primers, having non-homologous sequences to act as linker/primers,were prepared as follows:

A. Automated Synthesis Procedures: The diethyl phosphoramidites,synthesized according to Beaucage and Caruthers (Tetrahedron Letters(1981) 22:1859-1862) were sequentially condensed to a nucleosidederivatized controlled pore glass support using a Biosearch SAM-1. Theprocedure included detritylation with trichloroacetic acid indichloromethane, condensation using benzotriazole as activating protondonor, and capping with acetic anhydride and dimethylaminopyridine intetrahydrofuran and pyridine. Cycle time was approximately 30 minutes.Yields at each step were essentially quantitative and were determined bycollection and spectroscopic examination of the dimethoxytrityl alcoholreleased during detritylation.

B. Oligodeoxyribonucleotide Deprotection and Purification Procedures:The solid support was removed from the column and exposed to 1 mlconcentrated ammonium hydroxide at room temperature for four hours in aclosed tube. The support was then removed by filtration and the solutioncontaining the partially protected oligodeoxynucleotide was brought to55° C. for five hours. Ammonia was removed and the residue was appliedto a preparative polyacrylamide gel. Electrophoresis was carried out at30 volts/cm for 90 minutes after which the band containing the productwas identified by UV shadowing of a fluorescent plate. The band wasexcised and eluted with 1 ml distilled water overnight at 4° C. Thissolution was applied to an Altech RP18 column and eluted with a 7-13%gradient of acetonitrile in 1% ammonium acetate buffer at pH 6.0. Theelution was monitored by UV absorbance at 260 nm and the appropriatefraction collected, quantitated by UV absorbance in a fixed volume andevaporated to dryness at room temperature in a vacuum centrifuge.

C. Characterization of Oligodeoxyribonucleotides: Test aliquots of thepurified oligonucleotides were ³² p labeled with polynucleotide kinaseand γ-³² P-ATP. The labeled compounds were examined by autoradiographyof 14-20% polyacrylamide gels after electrophoresis for 45 minutes at 50volts/cm. This procedure verifies the molecular weight. Base compositionwas determined by digestion of the oligodeoxyribonucleotide tonucleosides by use of venom diesterase and bacterial alkalinephosphatase and subsequent separation and quantitation of the derivednucleosides using a reverse phase HPLC column and a 10% acetonitrile, 1%ammonium acetate mobile phase.

The above reaction mixtures were held in a heat block set at 95° C. for10 minutes to denature the DNA. Then each DNA sample underwent 28 cyclesof amplification, where each cycle was composed of four steps:

(1) spinning the sample briefly (10-20 seconds) in microcentrifuge topellet condensation and transfer the denatured material immediately to aheat block set at 30° C. for two minutes to allow primers and genomicDNA to anneal,

(2) adding 2 μl of a solution prepared by mixing 39 μl of the Klenowfragment of E. coli DNA Polymerase I (New England Biolabs, 5 units/μl),39 μl of a salt mixture of 100 mM Tris buffer (pH 7.5), 500 mM NaCl and100 mM MgCl₂, and 312 μl of water,

(3) allowing the reaction to proceed for two minutes at 30° C., and

(4) transferring the samples to the 95° C. heat block for two minutes todenature the newly synthesized DNA, except this reaction was not carriedout at the last cycle.

Then the mixtures were stored at -20° C. The following cloning procedurewas used for the amplified products.

The reaction mixture was sub-cloned into M13mp10 by first digesting in50 μl of a buffer containing 50 mM NaCl, 10 mM Tris.HCl, pH 7.8, 10 mMMgCl₂, 20 units PstI, and 26 units HindIII at 37° C. for 90 minutes. Thereaction was stopped by freezing. The volume was adjusted to 110 μl witha buffer containing Tris.HCl and EDTA and loaded onto a 1 ml BioGel P-4spin dialysis column. One fraction was collected and ethanolprecipitated.

The ethanol pellet was resuspended in 15 μl water and adjusted to 20 μlvolume containing 50 mM Tris.HCl, pH 7.8, 10 mM MgCl₂, 0.5 mM ATP, 10 mMdithiothreitol, 0.5 μg of M13mp10 vector digested with PstI and HindIIIand 400 units ligase. This mixture was incubated for three hours at 16°C.

Ten microliters of ligation reaction mixture containing Molt 4 DNA wastransformed into E. coli strain JM103 competent cells, which arepublicly available from BRL in Bethesda, Md. The procedure followed forpreparing the transformed strain is described in Messing, J. (1981)Third Cleveland Symposium on Macromolecules:Recombinant DNA, ed. A.Walton, Elsevier, Amsterdam, 143-153.

About 40 different alleles from these two cloning procedures weresequenced. In some of the sequences determined four areas of specificDNA and protein sequence were found to occur in various combinations andto be associated with IDDM. The DNA sequences seen in each of thesesegments in the genomes of IDDM patients produced an alteration in oneto three amino acid residues of the DRβ protein. These four variablesegments of the DRβ second exon, found in sequences obtained from manydiabetic sources, and labeled A-D, are identified above. The regionswhich can be used for devising probes used for detecting such sequencesare identified in FIG. 5, where the amino acid abbreviations are shownin Table VIII.

                  TABLE VIII                                                      ______________________________________                                        Amino Acid Abbreviation Codes                                                 ______________________________________                                        Alanine            Ala    A                                                   Arginine           Arg    R                                                   Asparagine         Asn    N                                                   Aspartic Acid      Asp    D                                                   Cysteine           Cys    C                                                   Glutamine          Gln    Q                                                   Glutamic Acid      Glu    E                                                   Glycine            Gly    G                                                   Histidine          His    H                                                   Isoleucine         Ile    I                                                   Leucine            Leu    L                                                   Lysine             Lys    K                                                   Methionine         Met    M                                                   Phenylalanine      Phe    F                                                   Proline            Pro    P                                                   Serine             Ser    S                                                   Threonine          Thr    T                                                   Tryptophan         Trp    W                                                   Tyrosine           Tyr    Y                                                   Valine             Val    V                                                   ______________________________________                                    

II. Preparation of Primers for Detection

Oligonucleotides designated GH46 and GH50 complementary to oppositestrands of the conserved 5' and 3' ends of the DR-β second exon wereused as primers. The primers, have the sequences identified in thesection above.

III. Expected Amplification Reaction

One microgram of DNA from each DNA sample to be tested (10 μl of 100μg/ml DNA) may be amplified in an initial 100 μl reaction volumecontaining 10 μl of a solution containing 100 mM Tris buffer (pH 7.5),500 mM NaCl, and 100 mM MgCl₂. 10 μl of 10 μM of primer GH46, 10 μl of10 μM of primer GH50, 15 μl of 40 mM dNTP (contains 10 mM each of dATP,dCTP, dGTP and TTP), 10 μl DMSO, and 45 μl of water.

Each reaction mixture is held in a heat block set at 95° C. for 10minutes to denature the DNA. Then each DNA sample undergoes 30 cycles ofamplification where each cycle is composed of four steps:

(1) spinning the sample briefly (10-20 seconds) in microcentrifuge topellet condensation and transfer the denatured material immediately to aheat block set at 37° C. for two minutes to allow primers and genomicDNA to anneal,

(2) adding 2 μl of a solution prepared by mixing 39 μl of the Klenowfragment of E. coli DNA Polymerase I (New England Biolabs, units/μl), 39μl of a salt mixture of 100 mM Tris buffer (pH 7.5), 500 mM NaCl and 100mM MgCl₂, and 312 μl of water,

(3) allowing the reaction to proceed for two minutes at 37° C., and

(4) transferring the samples to the 95° C. heat block for two minutes todenature the newly synthesized DNA, except this reaction was not carriedout at the last cycle.

The final reaction volume is 150 μl, and the reaction mixture is storedat -20° C.

IV. Expected Synthesis and Phosphorylation of OligodeoxyribonucleotideProbes

Two of four labeled DNA probes, designated GH54 (V-S) and GH78 (I-DE),from Regions C and D, respectively, are employed.

These two probes are synthesized according to the procedures describedabove for preparing primers for cloning. The probes are labeled bycontacting 10 pmole thereof with 4 units of T4 polynucleotide kinase(New England Biolabs) and about 40 pmole γ⁻³² P-ATP (New EnglandNuclear, about 7000 Ci/mmole) in a 40 μl reaction volume containing 70mM Tris buffer (pH 7.6), 10 mM MgCl₂, 1.5 mM spermine, 100 mMdithiothreitol and water for 60 minutes at 37° C. The total volume isthen adjusted to 100 μl with 25 mM EDTA and purified according to theprocedure of Maniatis et al., Molecular Cloning (1982), 466-467 over a 1ml Bio Gel P-4 (BioRad) Spin dialysis column equilibrated with Tris-EDTA(TE) buffer (10 mM Tris buffer, 0.1 mM EDTA, pH 8.0).

V. Expected Dot Blot Hybridizations

Five microliters of each of the 150 μl amplified samples from SectionIII was diluted with 195 μl 0.4 N NaOH, 25 mM EDTA and spotted ontothree replicate Genatran 45 (PLASCO) nylon filters by first wetting thefilter with water, placing it in a Bio-Dot (BioRad) apparatus forpreparing dot blots which holds the filter in place, applying thesamples, and rinsing each well with 0.4 ml of 20×SSPE (3.6M NaCl, 200 mMNaH₂ PO₄, 20 mM EDTA), as disclosed by Reed and Mann, Nucleic AcidsResearch, 13, 7202-7221 (1985). The filters are then removed, rinsed in20×SSPE, and baked for 30 minutes at 80° C. in a vacuum oven.

After baking, each filter is then contacted with 6 ml of a hybridizationsolution consisting of 5×SSPE, 5×Denhardt's solution (1×=0.02%polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin, 0.2 mMTris.HCl, 0.2 mM EDTA, pH 8.0) and 0.5% SDS and incubated for 60 minutesat 55° C. Then 5 μl each of the probes is added to the hybridizationsolution and the filters are incubated for 60 minutes at 55° C.

Finally, each hybridized filter is washed under stringent conditions.The genotypes are expected to be readily apparent after 90 minutes ofautoradiography. The probes are expected to have reasonable specificityfor the portions of the allele being detected in genomic DNA samples.

EXAMPLE II

Peptides to the amino acid sequences disclosed may be prepared asdescribed above and used as immunogens to generate antibodies thereto,useful in immunoassays for detecting the amino acid sequence(s) inprotein samples.

EXAMPLE III

To explore the possibility that both the DR4 and DRw6 haplotypes whichare associated with Pemphigus vulgaris (PV) contain a common epitopeindicative of PV susceptibility in the DRβI chain, the nucleotidesequences of the polymorphic second exon of the DRβ amd DQβ loci fromthree PV patients were determined. As the HLA-DR serotypes of the threePV patients were DR4/4, DR4/5 and DR4/5, only the issue of DR4associated PV susceptibility was explored in this example. The sequenceanalysis was carried out on M13 clones containing specific polymerasechain reaction (PCR) amplified fragments. [See Saiki et al., Science,230:1350-1354 (1985); and Scharf et al., Science, 233:1076-1078 (1986)for methodology associated with PCR amplification, cloning and sequenceanalysis.]

Sample Preparation and Amplification Procedures

Blood samples from three Pemphigus vulgaris patients were provided byDr. Bruce Wintroub, UCSF (California). 0.5 ml of whole blood was lysedby the addition of 1.5 ml of 10 mM Tris, pH 7.5, 10 mM EDTA, 100 mMNaCl, 40 mM dithiothreitol, and 200 μg/ml Proteinase K and incubated for16 hours at 55° C. The samples were phenol extracted, phenol-CHCl₃ andCHCl₃ extracted and ethanol precipitated overnight at -20° C. Theprecipitated DNA was pelleted by centrifugation, washed with 70%ethanol, dried and resuspended in 100 μl 10 mM Tris, 0.1 mM EDTAcontaining 100 μg/ml RNAse A and incubated at 37° C. for 15 minutes. TheDNA samples were re-extracted with phenol-CHCl₃ and CHCl₃ to inactivatethe RNAse A and ethanol precipitated, washed, and dried as describedabove. 1 μg of intact genomic DNA was amplied by polymerase chainreaction (PCR) (Scharf et al., id.) with the following changes in thereaction conditions: A.) The HLA DRβ region genes were amplified byusing 1 μM of the PCR primers GH46 and GH50 (See FIG. 1 for descriptionof primers and the HLA DRβ target fragment); 1 unit of cloned E. coliDNA polymerase 1 large fragment (Klenow fragment) was added for 20cycles of amplification; an additional five cycles of amplification wasrun on the samples using 4 units of Klenow fragment B.) The HLA DQβregion genes amplified by using 1 μM of PCR primers GH28 and GH29 (suchprimers are disclosed in copending, commonly owned U.S. application Ser.No. 899,344 filed Aug. 22, 1986):

GH28 (CTCGGATCCGCATGTGCTACTTCACCAACG)

GH29 (GAGCTGCAGGTAGTTGTGTCTGCACAC).

1 unit of Klenow fragment was added for 20 cycles of amplification; anadditional 8 cycles of amplification were carried out using 2 units ofKlenow fragment per cycle. The DQβ primers produce a 238 base-pairfragment. The DRβ primers produced a 272 bp fragment (see FIG. 1). 1/10of the PCR reactions were run on a 4% NuSieve, 0.5% SeaKem (FMC) agarosegel and transferred to Genatran 45 nylon membrane (Scharf et al., id.).The filter was prehybridized in 10 ml 5×SSPE, 4×Denhardt's and 0.5%sodium dodecyl sulfate (SDS) for 15 minutes at 37° C. The filter washybridized with the addition of 0.1 pmole of (γ-³² P) ATP labeled DRβspecific oligomer GH22 for 16 hours at 37° C. The filter was washed in4×SSPE, 0.1% SDS for 2.5 minutes at 30° C. and for 1.5 minutes at 37° C.and exposed for 16 hours at -70° C. with one intensifying screen (DuPontCronex Lightning Plus). The filter was stripped of probe by incubationfor five minutes in boiling 0.1×SSPE, 0.1% SDS, dried and prehybridizedin 10 ml 6×SSPE, 10×Denhardt's and 0.2% SDS at 42° C. 0.2 pmole of (γ-³²P) ATP labeled DR4 Dw10 sequence specific oligomer GH78 was added to theprehybridization solution and incubated for 16 hours at 42° C. Afterhybridization the filter was washed in 1×SSPE, 0.1% SDS for 10 minutesat 37° C. and autoradiographed.

Cloning and Seqencing of HLA DRβ and DQβ PCR Products.

One half of each PCR reaction was ethanol precipitated, resuspended andloaded onto a 4% NuSieve, 0.5% SeaKem gel and electrophoresed at 20V-cmfor one hour. Slices between 265 and 280 base-pairs for DRβ, and between220 and 250 base-pairs for DQβ, were removed from the gel and the DNAwas electroeluted from the gel in 200 μl 0.5×TBE buffer. Theelectroeluted DNA samples were dialyzed, digested with 60 units of BamHIand PstI (New England BigLabs) for three hours at 37° C., phenol,phenol-CHCl₃ and CHCl₃ extracted and ethanol precipitated. One-fourth ofthe digested PCR DNA was ligated to 200 μg of BamHI/PstI digested,dephosphorylated M13mp10 and transformed into E. coli JM103 and platedonto selective media [Scharf et al., id.; Messing, in Wu et al. (eds),Methods in Enzymology, 101, pp. 20-78 (1983)]. The positive clones wereidentified by in-situ plaque filter hybridization using anick-translated DRβ cDNA probe and plaque purified, and DNA from thepurified phage clones was sequenced by the chain termination method[Sanger et al., PNAS (U.S.A.), 74:5463-5468 (1977].

Analysis. Southern blot analysis of the PCR amplification products andof a DR-3 homozygous typing cell DNA (included as a general DRβamplification and hybridization control) using a DRβ cDNA probeindicated that all four samples contained roughly equal amounts ofamplified DR-beta fragment.

The filter was then stripped of the nick-translated DRβ probe andreprobed with GH78, an oligonucleotode probe specific for the DR4subtype Dw10 (see FIG. 1). The GH78 sequence specific oligonucleotide(SSO) hybridizes specifically to Dw10 sequences and to none of the otherDR4 subtypes (for example, Dw4, Dw14, Dw13 and Dw15). The DNA amplifiedfrom all three PV patients hybridized to GH78 but not to the DR-3homozygous control.

The amplified DNA fragments for the HLA DRβ and DQβ loci were cloned andsequenced to examine in more detail polymorphisms at the level of codingsequences. FIG. 2 shows amino acid alignments derived from nucleotidesequence data for HLA DRβ loci. The DR4 Dw10 subtype prototype sequenceis distinguished from the DR4 Dw4 prototype sequence by the substitutionof isoleucine, aspartic acid, and glutamic acid (the "I-DE" epitope) forleucine, glutamine, and lysine at amino acid positions 68, 71, and 72,respectively. The sequence alignments show that the DRβ sequences fromthe DR4 alleles of these three patients also contain this "I-DE"epitope). The DRβ sequences from the DR5 alleles from the two DR4/5patients (patients II and III) are identical to the DRβ1 sequence fromthe DR5 prototype. Likewise, the DRβIII sequences from the DR5 allelesfrom these two DR4/5 patients are identical to the DR5 DRβIII prototypesequence. Patient I, who is DR4/4, has one Dw10 and one Dw13 halotype,and the DRβIV sequence from this patient is identical to the DR4IVprototype sequence. The amino acid alignments derived from thenucleotide sequence data for the HLA DQβ loci are shown in FIG. 3.Patient I subtypes as DQB3.2 for one DR4 allele and DQB3.1 for the otherDR4 allele. Patients II and III (both DR4/5) are also heterozygous fortheir DQβ loci; the DR4 allele is DQB3.2 and the DQB3.1 locus is fromthe DR5 haplotype. Given the DR serotypes, this heterozygosity at theDQβ locus is not surprising since both DQB3.1 and DQB3.2 are associatedwith the DR4 haplotype and DQB3.1 is associated with the DR5 halotype.The DQB3.1 and DQB3.2 sequences from all three patients are identicalwith their respective prototype sequences.

EXAMPLE IV

The same sequence near codon 70 described above in Example III thatdistinguishes DR4-Dw10 from the other DR4-Dw subtypes is also present ona subset of DRw6 haplotypes. The pattern of sequence polymorphismsuggested that this shared "epitope" could be responsible for the DR4and DRw6 disease association with PV.

DNA samples from Israeli patients, DR-matched controls, and Austrianpatients were analyzed with a panel of DRβ and DQβ sequence-specificoligonucleotide probes using PCR amplified DNA in a dot blot format asdescribed above. In the analysis with DRβ-1 oligonucleotide probes,essentially all of the patient DR4 haplotypes (24/24 Israeli patients;10/14 non-Israeli patients) had the Dw10 associated epitope versus asmaller proportion of the control DR4 haplotypes (15/25 Israelicontrols; 1/19 non-Israeli controls). However, the proporton of DRw6DRβ-I alleles that contain that epitope was lower in DRw6 PV patients(4/14) relative to controls (8/13). Therefore, it was concluded that ifthe DR4 susceptibility to PV can be attributed to a specific DRβ1allele, then the DRw6 susceptibility must be accounted for by adifferent sequence.

EXAMPLE V PCR Amplification of DQβ Genomic Sequences.

To obtain the results shown in FIG. 4, 1 μg of genomic DNA from each ofthe various cell lines shown in the left-hand side of FIG. 4 wasamplified by PCR [Saiki et al., Nature, 324:163-166 (1986)] for 28cycles with the Klenow fragment of E. coli DNA polymerase. Theamplification primers GH28 and GH29 (see Example IV) were used toamplify a 238-bp segment of the DQβ gene. The degree and specificity ofamplification were monitored by agarose gel electrophoresis, blotting,and hybridization with 32P-labeled DQβ cDNA probes (Scharf et al.,supra). The sources of the IDDM patients are described in Arnheim etal., supra.

PCR Cloning into M13mp10

The amplified DNA was extracted once with an equal volume ofTE-saturated phenol, followed by two extractions with aphenol/chloroform mixture. The DNA was then diluted to 2 ml with sterilewater, and dialyzed and concentrated by centrifugation through aCentricon 10 column (Amicon) at 5000×g for 60 minutes at roomtemperature. In the case of DQβ amplification, where the PCR primersproduce a number of non-HLA amplification products, the target band wasfirst cut out of an agarose gel and electroeluted at 100 volts for onehour in 0.5×TBE. The DNA was diluted to 100 μl and digested for twohours at 37° C. with 40 units each of BamHI and PstI. After digestion,the DNA was again phenol/chloroform extracted, dialyzed, andconcentrated. It was then ligated into the M13mp10 vector by amodification of the PCR-cloning procedure (Scharf et al., supra). TheM13 plaques were initially screened for inserts by hybridization witheither DQβ or DQβ cDNA probes, and occasionally with allele-specificoligonucleotide (ASO) probes (Saiki et al., supra). These clones werealso screened for the amplification products of the DXα or DXβ genes bythe use of ASO probes. The single-strand phage DNA was isolated bystandard techniques, and sequenced by the dideoxynucleotide primerextension method [Smith, Meth. Enzymol., 65:560-580 (1980)].

EXAMPLE VI Viral Sequence Homology

The sequence of the 172282-bp genome of strain B95-8 of Epstein-Barrvirus (Baer et al., supra) was translated by computer into proteinsequences for all six of the forward and reverse phases. Thesetranslations were then searched for exact matches of five or more aminoacids with polypeptide sequences centered at position 57 of the DQβalleles (see Table VII). The location and size of the open reading frame(ORF) for each match was then determined, and correlated with the majorORF's and transcriptional segments of the EBV genome (Baer et al,supra). One method of judging the significance of these homologies is byestimating their chance of occurring at random. In the six translationphases of EBV, the amino acid residues near position 57 of the DQβalleles occur at fractions: A: 0.0804, D=0.0258, E=0.0337, G=0.1089,L=0.0890, P=0.1089, R=0.0962, and Y=0.0141. By multiplying thesefrequencies together, the chances of random occurrence for thepolypeptide epitopes are: "RPDAE"=1/1,370,000, "GLPAA"=1/147,000,"PAAEY"=1/2,990,000, "GPPAA"=1/120,000 and "PPAAEY"=1/27,400,000. Thus,since there are 344,560 residues in all six of the translations, wewould expect several random occurrences for the "GPPAA" and "GLPAA"epitopes, but none for the others. The excess number of matches andtheir correlation with major ORF's and with repeated segments of thegenome contribute to their significance. By statistical probabilitiesalone, the six-residue match "PPAAEY" is particularly unlikely to haveoccurred at random.

Other modifications of the above described embodiments of the inventionthat are obvious to those skilled in the area of molecular and clinicalbiology and diagnostics and related disciplines are intended to bewithin the scope of the following claims.

What is claimed is:
 1. A process for detecting if a sequence associatedwith susceptibility to insulin dependent diabetes mellitus (IDDM) orDR4-associated Pemphigus vulgaris is present in a DNA sample,comprising:(a) exposing said DNA sample to an oligonucleotide probeunder stringent hybridization conditions, wherein said probe is fifteento twenty-five nucleotides in length and complementary to a sequence inthe second exon of a class II HLA-β-chain gene, wherein said probedifferentiates between alleles present at a higher frequency inindividuals susceptible to insulin dependent diabetes mellitus (IDDM) orDR4-associated Pemphigus vulgaris and alleles found at higher frequencyin non-susceptible individuals, wherein said probe comprises a sequenceselected from the group of sequences consistingof5'-GACATCCTGGAAGACGAGC-3', 5'-GACATCCTGGAAGACGAGCGG-3', and DNAsequences fully complementary thereto; and (b) detecting whether saidprobe has hybridized to said DNA sample.
 2. A process of claim 1,wherein said probe comprises a sequence 5'-GACATCCTGGAAGACGAGC-3', or aDNA sequence fully complementary thereto.
 3. A process of claim 2,wherein said probe is 5'-GACATCCTGGAAGACGAGC-3', or a DNA sequence fullycomplementary thereto.
 4. A process of claim 1, wherein said probedifferentiates between alleles present at a higher frequency inindividuals susceptible to DR4 Dw10-associated Pemphigus vulgaris andalleles found at higher frequency in non-susceptible individuals, andwherein said probe comprises the sequence 5'-GACATCCTGGAAGACGAGCGG-3',or a DNA sequence fully complementary thereto.
 5. A process of claim 4,wherein said probe is 5'-GACATCCTGGAAGACGAGCGG-3', or a DNA sequencefully complementary thereto.
 6. A process for detecting if a sequenceassociated with susceptibility to DRw6-associated Pemphigus vulgaris ispresent in a DNA sample, comprising:(a) exposing said DNA sample to anoligonucleotide probe under stringent hybridization conditions, whereinsaid probe is fifteen to twenty-five nucleotides in length andcomplementary to a sequence in the second exon of a class II HLA-β-chaingene, wherein said probe differentiates between alleles present at ahigher frequency in individuals susceptible to DRw6-associated Pemphigusvulgaris and alleles found at higher frequency in non-susceptibleindividuals; and (b) detecting whether said probe has hybridized to saidDNA sample.
 7. A process of claim 6, wherein said probe comprises anucleotide sequence from a second exon of a DQB1.3 gene in the regionfrom codon 20 to codon
 80. 8. A process for detecting if a DR3 beta-IIIDNA sequence associated with susceptibility to insulin dependentdiabetes mellitus (IDDM) is present in a DNA sample, comprising:(a)exposing said DNA sample to an oligonucleotide probe under stringenthybridization conditions, wherein said probe is fifteen to twenty-fivenucleotides in length and complementary to a sequence in the second exonof a class II HLA-β-chain gene, and wherein said probe comprises asequence selected from the group consisting of:5'-GAGCTGCGTAAGTCTGAG-3',5'-GAGGAGTTCCTGCGCTTC-3', 5'-CCTGTCGCCGAGTCCTGG-3', and sequences fullycomplementary thereto; and (b) detecting whether said probe hashybridized to said DNA sample.
 9. A process of claim 8, wherein saidprobe consists of a sequence selected from the group consistingof:5'-GAGCTGCGTAAGTCTGAG-3', 5'-GAGGAGTTCCTGCGCTTC-3',5'-CCTGTCGCCGAGTCCTGG-3', andsequences fully complementary thereto. 10.The process of claim 6, wherein said probe comprises a sequence selectedfrom the group consisting of:5'-TCGGCGTCAGGCCGCCCCT-3',5'-TCGGCAACAGGCCGCCCCT-3', andsequences fully complementary thereto. 11.The process of claim 10, wherein said probe consists of a sequenceselected from the group consisting of:5'-TCGGCGTCAGGCCGCCCCT-3',5'-TCGGCAACAGGCCGCCCCT- 3', andsequences fully complementary thereto.